我们感谢张庆信教授对缺氧工作站的使用。这项工作得到以下方面的支持: 新加坡教育部、教育部 1T1-02/04 和 MOE2015-T2-2-087 (Y.A.)、李港医学院、南洋理工大学开办补助金 M4230003 (P.O.B.)、瑞典研究理事会、家庭苓基金会, 诺德基金会, Stichting af Jochnick 基金会, 瑞典糖尿病协会, 氧化钪保险公司, 糖尿病研究和健康基金会, 泊位冯 Kantzow 的基础,战略研究计划在糖尿病卡罗林斯卡 Institutet, 紧急救济 ERC-2013-AdG 338936 Betalmage, 和克努特和爱丽丝拉乌尔·沃伦贝格基金会。

本协议部分采用人类胚胎肾脏 293A (HEK293A) 细胞, 遵循新加坡南洋理工大学人类研究伦理学委员会的指导方针。
1. HEK293A 细胞缺氧的诱导
<ol><li>准备四10厘米的菜肴和种子 3–5 x 106 HEK293A 细胞每盘在10毫升 Dulbecco 的改良鹰的培养基 (DMEM, 4.5 克/升葡萄糖) 辅以10% 胎牛血清 (血清), 2 毫米 l-谷氨酰胺, 110 毫克/升丙酮钠, 100 U/毫升青霉素和100毫克/链霉素。培养细胞在37°c, 5% CO2孵化器。 注: 细胞播种应在生物安全柜 (BSC) 进行。要放置在平衡计分卡上的所有材料的表面应用70% 乙醇擦拭。</li>
	<li>24小时播种后, 当细胞达到80–90% 融合时, 将两个盘子放入孵化 glovebox 中的缺氧 subchamber (见材料表) 与 1% O2和 5% CO2在37°c, 并保持其他两个盘子在 normoxia (21% O2, 5% co2在37°c) 为 4 h. 利用一组常氧和缺氧细胞对免疫沉淀的低氧治疗进行评价, 并利用另一组细胞进行联合实验。 注: 氧水平可以设定在 0–5%, 缺氧治疗的持续时间可以根据细胞类型和研究目标而变化。</li>
</ol>2. 全细胞和核萃取
注: 有关此协议中使用的缓冲区的信息, 请参见表 1 。
<ol><li>
收获 normoxia 控制细胞。
	<ol>
<li>用10毫升 DPBS (PH 7.0–7.2) 用10毫升的吸管去除培养基, 用吸入法冲洗细胞。 注意: 避免用吸管接触细胞单层。在洗涤过程中, 轻轻地将 DPBS 在细胞培养板的壁上, 以避免细胞流失。</li>
		<li>吸管5毫升冰冷 DPBS 进入板和刮的细胞表面上的冰冷 PBS 与细胞刮刀。</li>
		<li>将细胞悬浮液转移到15毫升锥形管中, 并保持在冰上。</li>
	</ol>
</li>
	<li>
在缺氧条件下收获细胞培养。
	<ol>
<li>预平衡的 DPBS 到缺氧条件通过放置一个发现100 毫升实验玻璃试剂瓶填充 DPBS 在缺氧 subchamber (1% O2和 5% CO 2 在 37 °c), 提前24小时。</li>
		<li>大约 1 h 在收获缺氧治疗的细胞之前, 放置一个冰盒包含冰入 glovebox 的处理室, 已经平衡到 1% O2和 5% CO2。将含有预平衡缺氧 DPBS 的瓶子从缺氧 subchamber 转移到处理室并放在冰上。</li>
		<li>低氧治疗后4小时, 将细胞从缺氧 subchamber 转移到平衡前 1% O2和 5% CO2的处理室。</li>
		<li>通过吸入去除培养基, 用10毫升冰冷预平衡缺氧 DPBS 和10毫升吸管冲洗细胞一次。</li>
		<li>添加5毫升冰冷预平衡 DPBS 与5毫升吸管, 并通过刮一个细胞刮刀去除细胞。</li>
		<li>倾斜细胞培养板和收集分离细胞使用10毫升吸管。将 DPBS 中的细胞悬浮液转移到15毫升锥形管中, 并保持在冰上。</li>
		<li>打开 glovebox 的处理和缓冲室之间的门, 两者都已预平衡到 1% O2 和 5% CO2。将含有缺氧处理细胞的冰上的15毫升锥形管转移到缓冲室。打开缓冲室的门, 完全从 glovebox 中取出细胞。</li>
	</ol>
</li>
	<li>常氧细胞从2.1 和缺氧细胞从2.2 通过离心在 1000 x g为5分钟在4°c。</li>
	<li>
准备整个细胞提取物。
	<ol>
<li>并用重悬500µL 中的细胞颗粒, 包括50毫米 Trisaminomethane 盐酸盐 (三盐酸) pH 8.0、150毫米氯化钠 (NaCl)、1% tergitol 型 NP-40 (NP-40)、0.5% 钠脱氧和0.1% 月桂酸钠 (SDS), 辅以1x 蛋白酶抑制剂鸡尾酒 (见材料表) 通过吹打细胞颗粒上下几次。</li>
		<li>将细胞裂解物转移到新的1.5 毫升离心管中, 并在冰上保持30分钟, 偶尔涡流。</li>
		<li>离心细胞裂解物在 1.3万 x g 10 分钟在4°c。</li>
		<li>收集上清, 整除50µL 的上清到1.5 毫升离心管, 并存储在-80 摄氏度。</li>
	</ol>
</li>
	<li>
用核萃取试剂盒准备核提取物 (见材料表)。
	<ol>
<li>用1x 蛋白酶抑制剂鸡尾酒和0.1 米 dithiothreitol (并用重悬) 在500µL 的裂解缓冲液中轻轻地将细胞颗粒向上和向下吹打几次。</li>
		<li>在十年代以最大速度添加25µL 的洗涤剂溶液 NP 到细胞悬浮和漩涡。</li>
		<li>离心机在 1万 x g 5 分钟在4摄氏度。</li>
		<li>收集上清 (细胞质提取物), 整除50µL 的上清到1.5 毫升离心管, 并贮存在-80 摄氏度。</li>
		<li>并用重悬500µL 裂解缓冲物中含有细胞核的球团, 辅以1x 蛋白酶抑制剂鸡尾酒和0.1 米的涡流, 最大速度为5秒。</li>
		<li>离心机在 1万 x g 5 分钟在4°c 和保存核球团。</li>
		<li>并用重悬50µL 萃取缓冲液中的核球团 NX1 辅以1x 蛋白酶抑制剂鸡尾酒, 多次吹打颗粒。</li>
		<li>在冰上孵化30分钟, 涡流为十年代每5分钟在最大速度。</li>
		<li>离心机在 1.2万 x g 10 分钟在4摄氏度。</li>
	</ol>
</li>
	<li>
淡化核萃取物。
	<ol>
<li>从步骤2.5.9 收集上清液, 并转入微型透析装置, 最大容积为每单位100µL。</li>
		<li>将微型透析装置盖上, 放在浮选装置中。</li>
		<li>将浮选装置放在装有500毫升预冷透析缓冲液的烧杯中 (20mM 三盐酸 pH 7.4, 20% 甘油, 100mM 氯化钾 (氯化钾), 0.2 毫米乙二胺二乙酸 (EDTA), 0.2 毫米 phenylmethylsulfonyl 氟 (PMSF) 和0.5 毫米)并孵育30分钟, 在4摄氏度与温和搅拌。 警告: PMSF 是危险的。避免直接接触皮肤或吸入。</li>
		<li>收集的样品从角落的迷你透析设备和转移到新的1.5 毫升离心管。</li>
		<li>离心机在 1.2万 x g 10 分钟在4°c, 整除25µL 每上清入1.5 毫升离心管, 并且存放在-80 °c。</li>
	</ol>
</li>
</ol>3. HIF-1α蛋白表达和亚细胞定位评价低氧治疗的价值
<ol><li>根据制造商的说明18, 测定二辛可宁酸 (微板块) 蛋白检测试剂盒的全细胞或核/细胞质提取物的蛋白质浓度。</li>
	<li>稀释细胞裂解物在 1x Laemmli 样品缓冲包含 5% 2-巯基乙醇并且煮沸在95°c 为5分钟。 注意: 不要接触加热块的表面, 因为它可能会引起烧伤。</li>
	<li>
用月桂酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 分离蛋白质。
	<ol>
<li>将等量的蛋白质 (25 µg) 加载到 SDS 页预制梯度凝胶 (4–20%) 的每个井中, 以及分子量标记的3µL。</li>
		<li>运行的凝胶在运行缓冲区中含有2.5 毫米三, 19.2 毫米甘氨酸, 0.01% SDS, PH8.3 30 分钟 200 v。</li>
	</ol>
</li>
	<li>
将蛋白质从凝胶转移到硝化膜。
	<ol>
<li>将转移夹层 (过滤纸-凝胶膜过滤纸) 与阳极边上的凝胶和卡带负极端的膜组装起来。</li>
		<li>将卡带放入装有2.5 毫米 trisaminomethane (三)、19.2 mm 甘氨酸和20% 甲醇的转移缓冲器的转储罐中。 注意: 甲醇是易燃的, 应储存在易燃液体贮存柜内。</li>
		<li>在冷室的100伏处进行1小时的转移。</li>
	</ol>
</li>
	<li>在10毫升的阻挡缓冲器中, 用50毫米三氯 (pH 7.6), 150 毫米氯化钠, 0.1 吐温20和5% 个非脂牛奶, 在室温下在振动筛上阻挡印迹。</li>
	<li>用 anti-HIF-1α抗体 (1/500 稀释) 在4摄氏度的同一阻塞缓冲液中孵化出印迹。</li>
	<li>每次用50毫升的3次清洗污点5分钟。</li>
	<li>用辣根过氧化物酶 (HRP)-共轭二级抗体 (1/1000 稀释) 在室温下用10毫升含5% 非脂肪干牛奶的1小时孵化出印迹。</li>
	<li>每次用50毫升的3次清洗污点5分钟。</li>
	<li>将500µL 的增强 chemilumescent (ECL) 试剂 a 和 B 混合在1.5 毫升离心管和涡流中。</li>
	<li>在室温下将 ECL 基底涂抹在印迹上, 孵育1分钟。</li>
	<li>
利用电荷耦合器件 (CCD) 摄像成像系统捕获化学发光信号。
	<ol>
<li>通过接触膜的边缘与纸巾, 将膜放在板料保护器中, 排出多余的 ECL 基体。</li>
		<li>将膜放在 CCD 摄像成像系统的样品托盘上。</li>
		<li>启动图像处理软件 (参见材料表), 并使用以下设置捕获图像: 文件→新协议→单通道→协议设置→凝胶成像 (应用: 化学;成像区域: 生物 Rad 准备凝胶;成像曝光: 软件将自动优化的曝光时间为强频带) →运行协议 注意: 可以手动设置曝光时间, 以实现最佳图像。</li>
	</ol>
</li>
</ol>4. Immunoprecipitated 蛋白的免疫沉淀和检测
<ol><li>在1.5 毫升离心管中, 将50µL 的蛋白 A/G 琼脂糖珠在500µL 的 tb 缓冲器中洗净, 用离心法在 3000 x G上以2°c 的方法将珠子颗粒化。</li>
	<li>在100µL 的 tb 缓冲器中, 丢弃上清和并用重悬珠子。</li>
	<li>添加2µL 的小鼠单克隆抗 ARNT 抗体 (1.4 毫克/毫升) 或小鼠免疫球蛋白 G (IgG) (1.4 毫克/毫升), 通过重组0.7 毫克鼠 IgG 在500µL tb 缓冲。</li>
	<li>放置1.5 毫升的离心管, 其中包含珠在管转子和孵化2小时在 10 rpm 在冷室。 注意: 轻轻地处理管子, 并保持悬浮在管子底部的珠子。</li>
	<li>以 3000 x g为2分钟, 在4摄氏度处离心成珠, 然后丢弃上清液。</li>
	<li>稀释200µg 在步骤2.6.5 中获得的核蛋白裂解物, 其800µL 的 IP 缓冲器由50毫米三盐酸 (pH 7.4)、180毫米氯化钠、20% 甘油、0.2% NP-40 和1x 蛋白酶抑制剂鸡尾酒组成。在4摄氏度, 在步骤4.5 中获得的抗体耦合珠孵化出裂解液。</li>
	<li>以 3000 x g为2分钟, 在4摄氏度时, 将珠子颗粒离心, 丢弃上清液, 用1毫升冰冷 tb (含 0.2% NP-40) 将珠子洗净3次。</li>
	<li>将珠子煮沸50µL Laemmli 样品缓冲器95°c 5 分钟。</li>
	<li>离心 1万 x g 5 分钟的珠子在4摄氏度, 收集上清, 并丢弃珠。 注: 上清液可储存在4摄氏度, 短期或-20 摄氏度长期。</li>
	<li>从 immunoprecipitated 蛋白配合物中检测出 HIF-1α的存在, 如上文步骤3.3 所述。 注: 此步骤不需要蛋白质量化。将每个样品上清的全部容积 (50 µL) 装入 SDS 页预制梯度凝胶 (4–20%) 的每个井中。</li>
</ol>

<ol>
	<li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factors+in+physiology+and+medicine.">Hypoxia-inducible factors in physiology and medicine.</a> Cell. 148 (3), 399-408 (2012).</li><li>Bartels, K., Grenz, A., Eltzschig, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+inflammation+are+two+sides+of+the+same+coin.">Hypoxia and inflammation are two sides of the same coin.</a> Proc Natl Acad Sci U S A. 110 (46), 18351-18352 (2013).</li><li>Jiang, B. H., Rue, E., Wang, G. L., Roe, R., Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerization,+DNA+binding,+and+transactivation+properties+of+hypoxia-inducible+factor+1.">Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1.</a> J Biol Chem. 271 (30), 17771-17778 (1996).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1:+mediator+of+physiological+and+pathophysiological+responses+to+hypoxia.">HIF-1: mediator of physiological and pathophysiological responses to hypoxia.</a> J Appl Physiol. 88 (4), 1474-1480 (2000).</li><li>Kallio, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+in+hypoxic+cells:+Inducible+nuclear+translocation+and+recruitment+of+the+CBP/p300+coactivator+by+the+hypoxia-inducible+factor-1alpha.">Signal transduction in hypoxic cells: Inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha.</a> EMBO J. 17 (22), 6573-6586 (1998).</li><li>Ke, Q., Costa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor-1+(HIF-1).">Hypoxia-inducible factor-1 (HIF-1).</a> Mol Pharmacol. 70 (5), 1469-1480 (2006).</li><li>Gustafsson, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+requires+notch+signaling+to+maintain+the+undifferentiated+cell+state.">Hypoxia requires notch signaling to maintain the undifferentiated cell state.</a> Dev Cell. 9 (5), 617-628 (2005).</li><li>Zheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+with+factor+inhibiting+HIF-1+defines+an+additional+mode+of+cross-coupling+between+the+Notch+and+hypoxia+signaling+pathways.">Interaction with factor inhibiting HIF-1 defines an additional mode of cross-coupling between the Notch and hypoxia signaling pathways.</a> Proc Natl Acad Sci U S A. 105 (9), 3368-3373 (2008).</li><li>D'Ignazio, L., Bandarra, D., Rocha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB+and+HIF+crosstalk+in+immune+responses.">NF-kappaB and HIF crosstalk in immune responses.</a> FEBS J. 283 (3), 413-424 (2016).</li><li>Wang, G. L., Jiang, B. H., Rue, E. A., Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor+1+is+a+basic-helix-loop-helix-PAS+heterodimer+regulated+by+cellular+O2+tension.">Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.</a> Proc Natl Acad Sci U S A. 92 (12), 5510-5514 (1995).</li><li>Zheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type-specific+regulation+of+degradation+of+hypoxia-inducible+factor+1+alpha:+Role+of+subcellular+compartmentalization.">Cell-type-specific regulation of degradation of hypoxia-inducible factor 1 alpha: Role of subcellular compartmentalization.</a> Mol Cell Biol. 26 (12), 4628-4641 (2006).</li><li>Depping, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+translocation+of+hypoxia-inducible+factors+(HIFs):+involvement+of+the+classical+importin+alpha/beta+pathway.">Nuclear translocation of hypoxia-inducible factors (HIFs): involvement of the classical importin alpha/beta pathway.</a> Biochim Biophys Acta. 1783 (3), 394-404 (2008).</li><li>Wei, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+induces+oncogene+yes-associated+protein+1+nuclear+translocation+to+promote+pancreatic+ductal+adenocarcinoma+invasion+via+epithelial-mesenchymal+transition.">Hypoxia induces oncogene yes-associated protein 1 nuclear translocation to promote pancreatic ductal adenocarcinoma invasion via epithelial-mesenchymal transition.</a> Tumour Biol. 39 (5), (2017).</li><li>Chang, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+promotes+nuclear+translocation+and+transcriptional+function+in+the+oncogenic+tyrosine+kinase+RON.">Hypoxia promotes nuclear translocation and transcriptional function in the oncogenic tyrosine kinase RON.</a> Cancer Res. 74 (16), 4549-4562 (2014).</li><li>Moriya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+nature+of+overexpression+experiments.">Quantitative nature of overexpression experiments.</a> Mol Biol Cell. 26 (22), 3932-3939 (2015).</li><li>Prelich, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+overexpression:+Uses,+mechanisms,+and+interpretation.">Gene overexpression: Uses, mechanisms, and interpretation.</a> Genetics. 190 (3), 841-854 (2012).</li><li>Zheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Notch-independent+mechanism+contributes+to+the+induction+of+Hes1+gene+expression+in+response+to+hypoxia+in+P19+cells.">A Notch-independent mechanism contributes to the induction of Hes1 gene expression in response to hypoxia in P19 cells.</a> Exp Cell Res. 358 (2), 129-139 (2017).</li><li>Farris, M. H., Ford, K. A., Doyle, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+and+quantitative+assays+for+detection+and+characterization+of+protein+antimicrobials.">Qualitative and quantitative assays for detection and characterization of protein antimicrobials.</a> J Vis Exp. (110), e53819 (2016).</li><li>Chilov, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+nuclear+translocation+of+hypoxia-inducible+factor-1+(HIF-1):+heterodimerization+with+ARNT+is+not+necessary+for+nuclear+accumulation+of+HIF-1alpha.">Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1alpha.</a> J Cell Sci. 112 (Pt 8), 1203-1212 (1999).</li><li>Yin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arylsulfonamide+KCN1+inhibits+in+vivo+glioma+growth+and+interferes+with+HIF+signaling+by+disrupting+HIF-1alpha+interaction+with+cofactors+p300/CBP.">Arylsulfonamide KCN1 inhibits in vivo glioma growth and interferes with HIF signaling by disrupting HIF-1alpha interaction with cofactors p300/CBP.</a> Clin Cancer Res. 18 (24), 6623-6633 (2012).</li><li>Holmquist-Mengelbier, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+HIF-1alpha+and+HIF-2alpha+to+common+target+genes+is+differentially+regulated+in+neuroblastoma:+HIF-2alpha+promotes+an+aggressive+phenotype.">Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype.</a> Cancer Cell. 10 (5), 413-423 (2006).</li><li>Koh, M. Y., Powis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passing+the+baton:+The+HIF+switch.">Passing the baton: The HIF switch.</a> Trends Biochem Sci. 37 (9), 364-372 (2012).</li><li>Dumetz, A. C., Snellinger-O'brien, A. M., Kaler, E. W., Lenhoff, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+protein+protein+interactions+in+salt+solutions+and+implications+for+protein+crystallization.">Patterns of protein protein interactions in salt solutions and implications for protein crystallization.</a> Protein Sci. 16 (9), 1867-1877 (2007).</li><li>Graven, K. K., Troxler, R. F., Kornfeld, H., Panchenko, M. V., Farber, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+endothelial+cell+glyceraldehyde-3-phosphate+dehydrogenase+expression+by+hypoxia.">Regulation of endothelial cell glyceraldehyde-3-phosphate dehydrogenase expression by hypoxia.</a> J Biol Chem. 269 (39), 24446-24453 (1994).</li><li>Caradec, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desperate+house+genes':+The+dramatic+example+of+hypoxia.">Desperate house genes': The dramatic example of hypoxia.</a> Br J Cancer. 102 (6), 1037-1043 (2010).</li></ol>

作者声明没有利益冲突。

HIF-1 复合体是细胞氧稳态的主调节器, 它调节了多种细胞适应缺氧反应的基因。新的 HIF-1 相互作用蛋白的鉴定对于了解缺氧信号转导具有重要意义。免疫沉淀实验通常用于 PPIs 研究, 描绘细胞信号转导通路。然而, 蛋白质过度表达仍然被广泛使用, 这可能导致实验性工件。此外, HIF-1α是一种高度不稳定的蛋白质, 它在重氧11期间迅速降解。此外, 缺氧会触发大多数哺乳动物细胞系中 HIF-1 成分的核易位。因此, 必须对常规的免疫沉淀协议进行优化, 以确定缺氧治疗后生理相关的 HIF-1 相互作用蛋白。在这里, 我们描述了一个改进的免疫沉淀协议, 用来证明 HIF-1α和 ARNT 在缺氧之间的相互作用。
为避免 HIF-1α在再氧过程中的降解, 在 glovebox 的培养室中, 缺氧细胞在平衡缺氧条件下被收获。DPBS 洗涤缓冲器在使用前也平衡缺氧条件。如果没有缺氧工作站可供加工, 则可以用预平衡缺氧 DPBS 清洗缺氧细胞, 然后在冰上迅速收获。考虑到缺氧可触发 HIF-1 成分的核易位, 而蛋白过度表达可能诱发 artefactual 结果, 本协议使用内源核蛋白。其他20人也报告了在共同免疫沉淀议定书中使用核萃取物的情况。它代表了研究核蛋白之间相互作用的技术优势, 因为它最大限度地减少了核蛋白被全细胞蛋白稀释的几率。此外, 使用核萃取物可能有助于减少非特异性的相互作用, 特别是从富含大量无关的细胞质蛋白, 并通过最大限度地减少接触到核蛋白的稳定性细胞质中存在蛋白酶。使用内源核萃取物代替全细胞提取物对免疫沉淀显示了显著的技术改进, 因为我们只能检测 HIF-1α和 GABPα使用内源核萃取物之间的相互作用, 但不使用整体细胞提取物17。
在描述的协议中, 可以进一步优化几个条件以提高结果。以1% 氧为4小时, 诱导 HEK293A 细胞缺氧。但是氧水平和缺氧治疗的持续时间可以不同的细胞类型, 并根据研究的目的进行调整。例如, HIF-1α和 HIF-2α可以在细胞类型的特定方式下对缺氧进行差异调节, 表明它们在不同生物学环境中的独特作用。结果表明, 在神经母细胞瘤中, HIF-1α在短时间的剧烈缺氧 (1% o2) 时最活跃, 而 HIF-2α在轻度缺氧 (5% o2) 下活跃, 在慢性缺氧治疗后变得更加活跃 (48–72缺氧暴露)21。0–5% o2水平通常用于体外缺氧治疗, 其中 1–5% o2, 0.1–1%2和 0–0.1% o2经常被定义为轻度缺氧, 缺氧和缺氧, 分别为22。盐浓度是另一个需要优化的参数, 特别是因为它在 PPIs23中对离子相互作用起着关键作用。在这项研究中使用核萃取物, 通常用含有高浓度盐的缓冲液提取核蛋白。因此, 在共同免疫沉淀实验之前, 核萃取物可能必须淡化。在这里, 我们用含有100毫米氯化钾的透析缓冲器淡化核萃取物, 并将透析提取物与含有180毫米氯化钠的 IP 缓冲器混合, 以达到接近150毫米的最终盐浓度, 这与生理细胞内 PPIs 条件。最后的盐浓度可以根据 PPIs 的性质进行修改。在本协议中, GAPDH 被用作整个细胞提取物和细胞质组分的加载控制。我们没有观察到任何明显的缺氧诱导调节作用的 GAPDH 表达 HEK293A 细胞。然而, GAPDH 以前被证明是上调的缺氧在某些细胞类型24。有了这一点, 你可能需要使用替代适当的加载控制时, 必要的25。另外, 我们观察到在当前协议中使用的珠子或抗体所产生的大量背景信号。为了减少背景, 你可以延长洗涤步骤 (10–15分钟) 或执行超过3洗涤 (5–10时间)。另外, 洗涤缓冲液的离子强度也可以通过在洗涤过程中滴定盐浓度从150到500毫米来增加。裂解物也可以预先清除通过孵化与蛋白 A/G 琼脂糖珠1小时在4°c 与旋转。
本议定书仅限于核萃取物, 因此可能不适用于研究其他细胞室, 如线粒体和内质网中的 PPIs。类似于其他常规的免疫沉淀协议, 此方法不能用于实时研究 PPIs, 也不能用于确定 PPIs 是直接的还是间接的。
总之, 我们提供了一个改进的共同免疫沉淀协议, 以确定新的生理相关的 HIF-1 相互作用的蛋白质。该协议也适用于研究在缺氧条件下转录因子与转录联合调节剂之间的相互作用。虽然该协议是专门为在缺氧条件下进行的免疫沉淀实验而设计的, 但所描述的 normoxia 控制单元协议的一部分也可用于研究 normoxia 条件下的核 PPIs。

缺氧发生时, 没有足够的氧气供应给细胞和组织的身体。它在各种生理和病理过程中起着关键作用, 如干细胞分化、炎症和癌症1、2。缺氧诱导因子 (HIFs) 功能, heterodimers 由氧调节α亚基和组成性表达β亚基也称为 ARNT3。迄今已确定了 HIF α亚基 (HIF-1α、HIF-2α和 HIF-3α) 和三个 HIF β亚基 (ARNT/HIF-1β、ARNT2 和 ARNT3) 的三种异构体。HIF-1α和 ARNT 是无所不在表达的, 而 HIF-2α、HIF-3α、ARNT2 和 ARNT3 有更限制性的表达模式4。HIF-1 蛋白复合物是缺氧反应的关键调节因子。在缺氧条件下, HIF-1α变稳定, translocates 至细胞核, dimerizes ARNT5。随后, 这一复杂的结合到特定的核苷酸称为缺氧反应元素 (HREs), 并调节所涉及的目标基因的表达, 包括血管生成, 红细胞和糖酵解6。除了这种 "规范" 的反应, 缺氧信号通路也被称为串扰多细胞反应信号通路, 如缺口和核因子-卡伯 B (NF-nf-κb)7,8,9。
新的 HIF-1 相互作用蛋白的鉴定对于更好地理解缺氧信号通路是很重要的。与 ARNT, 这是不敏感的氧气水平和组成性表达, HIF-1α蛋白质水平是严格调节细胞氧水平。在 normoxia (21% 氧), HIF-1α蛋白质迅速退化10,11。HIF-1α在 normoxia 的短半衰期为细胞提取物中蛋白质的检测以及 HIF-1α-interacting 蛋白的鉴定提供了具体的技术挑战。此外, 一些转录因子, 包括那些 HIF-1 复合体植物常常将进入细胞核的缺氧条件下12,13,14。目前用于 PPI 研究的大多数方法都是使用非生理的蛋白质过度表达来进行的。这种蛋白过度表达通过多种机制 (包括资源超载、化学计量失衡、混杂相互作用和通路调制15、16) 引起了不同的细胞缺陷。在 PPI 研究方面, 蛋白质过度表达可能导致假阳性, 甚至是假阴性, 结果取决于蛋白质的性质和功能的抗原蛋白。因此, 目前的 PPI 研究方法必须修改, 以揭示在缺氧条件下的生理相关 PPIs。我们以前已经证明了 HIF-1 和 Ets 家族转录因子遗传结合蛋白 (GABP) 在缺氧 P19 细胞中的相互作用, 这有助于Hes1启动子对缺氧17的反应。在这里, 我们描述了一个共同免疫沉淀协议, 研究 PPIs 之间的内源性核蛋白在缺氧条件下。HIF-1α与 ARNT 的相互作用表现为概念的证明。该协议适用于证明转录因子与转录联合调节器在缺氧条件下的相互作用, 包括但不限于 HIF-1 相互作用蛋白的鉴定。

<table border="0" cellpadding="0" cellspacing="0" width="803">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Material</td>
			<td style="width:161px;"></td>
			<td style="width:135px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">1.0 M Tris-HCl Buffer, pH 7.4&#160;</td>
			<td style="width:161px;">1st BASE</td>
			<td style="width:135px;">1415</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Protein A/G Sepharose beads</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:135px;">ab193262</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Natural Mouse IgG protein</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:135px;">ab198772</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">EDTA</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610729</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">2x Laemmli Sample Buffer</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610737</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">2-Mercaptoethanol</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610710</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Nitrocellulose Membrane &#160;&#160;</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1620112</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Blotting-Grade Blocker</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1706404</td>
			<td style="width:229px;">Non-fat dry milk for western blotting applications</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10x Tris Buffered Saline (TBS)</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1706435</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10% Tween 20</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610781</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10x Tris/Glycine/SDS</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610732</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10x Tris/Glycine Buffer&#160;</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610771</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Precision Plus Protein Dual Color Standards</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1610374</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Anti-rabbit IgG, HRP-linked Antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:135px;">7074</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Anti-mouse IgG, HRP-linked Antibody&#160;</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:135px;">7076</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">SignalFire ECL Reagent</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:135px;">6883</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td style="width:161px;">Corning</td>
			<td style="width:135px;">21-030-CV</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td style="width:161px;">Merck Millipore</td>
			<td style="width:135px;">52332</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">ARNT/HIF-1 beta Antibody&#160;</td>
			<td style="width:161px;">Novus Biologicals</td>
			<td style="width:135px;">NB100-124&#160;</td>
			<td style="width:229px;">Concentration: 1.4 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">HIF-1 alpha Antibody</td>
			<td style="width:161px;">Novus Biologicals</td>
			<td style="width:135px;">NB100-479</td>
			<td style="width:229px;">Concentration: 1.0 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">YY1 Antibody</td>
			<td style="width:161px;">Novus Biologicals</td>
			<td style="width:135px;">NBP1-46218</td>
			<td style="width:229px;">Concentration: 0.2 mg/mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Qproteome Nuclear Protein Kit</td>
			<td style="width:161px;">Qiagen</td>
			<td style="width:135px;">37582</td>
			<td style="width:229px;">Lysis buffer NL and Extraction Buffer NX1 are provied in the kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">GAPDH Antibody</td>
			<td style="width:161px;">Santa Cruz</td>
			<td style="width:135px;">sc-47724</td>
			<td style="width:229px;">Concentration: 0.2 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycerol (&#8805;99%)</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:135px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Potassium chloride</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:135px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">RIPA buffer</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:135px;">R0278</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Sodium Chloride (NaCl)</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:135px;">71376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">NP-40</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:135px;">127087-87-0</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM, 4.5 g/L glucose)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">11995065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Dithiothreitol (DTT)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">R0861</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Fetal Bovine Serum</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">10270106</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">HEK293A cell line</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">R70507</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Methanol&#160;</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">67-56-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Penicillin-Streptomycin</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">15140122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Pierce Protease Inhibitor Tablets&#160;</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">88660</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">23225</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">QSP gel loading tip&#160;</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">QSP#010-R204-Q-PK</td>
			<td>1-200 uL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Equipment/Instrument</td>
			<td style="width:161px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Thick Blot Filter Paper, Precut, 7.5 x 10 cm</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1703932</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:279px;">Mini-PROTEAN&#160;Tetra Vertical Electrophoresis Cell for Mini Precast Gels, with Mini Trans-Blot&#160;Module and PowerPac Basic Power Supply</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1658034</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">4&#8211;15% Mini-PROTEAN TGX Precast Protein Gels</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">4561083</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">ChemiDoc XRS+ System</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1708265</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">I-Glove</td>
			<td style="width:161px;">BioSpherix</td>
			<td style="width:135px;">I-Glove</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Synergy HTX Multi-Mode Microplate Reader&#160;</td>
			<td style="width:161px;">BioTek</td>
			<td style="width:135px;">BTS1LFTA</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Costar 5mL Stripette Serological Pipets</td>
			<td style="width:161px;">Corning</td>
			<td style="width:135px;">4487</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Costar 10mL Stripette Serological Pipets</td>
			<td style="width:161px;">Corning</td>
			<td style="width:135px;">4488</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Costar 25mL Stripette Serological Pipets</td>
			<td style="width:161px;">Corning</td>
			<td style="width:135px;">4251</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Corning 96-Well Clear Bottom Black Polystyrene Microplates</td>
			<td style="width:161px;">Corning</td>
			<td style="width:135px;">3631</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">15mL High Clarity PP conical Centrifuge Tubes</td>
			<td style="width:161px;">Corning</td>
			<td style="width:135px;">352095</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Small Cell Scraper</td>
			<td style="width:161px;">Corning</td>
			<td style="width:135px;">3010</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Gilson&#160;Pipetman L 4-pipettes kit&#160;</td>
			<td style="width:161px;">Gilson</td>
			<td style="width:135px;">F167370</td>
			<td style="width:229px;">P2, P20, P200, P1000 and accessories</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">1.5mL Polypropylene Microcentrifuge Tubes</td>
			<td style="width:161px;">Greiner Bio-One&#160;</td>
			<td style="width:135px;">616201</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">PIPETBOY acu 2 Pipettor</td>
			<td style="width:161px;">INTEGRA Biosciences</td>
			<td style="width:135px;">155 000&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Justrite Flammable Liquid Storage Cabinets</td>
			<td style="width:161px;">Justrite Manufacturing Co.</td>
			<td style="width:135px;">896000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Vortex mixer</td>
			<td style="width:161px;">Labnet</td>
			<td style="width:135px;">S0200</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">CO2 incubator</td>
			<td style="width:161px;">NuAire</td>
			<td style="width:135px;">NU-5820</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Orbital shakers</td>
			<td style="width:161px;">Stuart</td>
			<td style="width:135px;">SSL1</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Tube rotator SB3</td>
			<td style="width:161px;">Stuart</td>
			<td style="width:135px;">SB3</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">MicroCL 21R Microcentrifuge</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">75002470</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Sorvall ST 16 Centrifuge</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">75004240</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Tissue Culture Dishes (100 mm)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">150350</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Slide-A-Lyzer MINI Dialysis Device</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">69580</td>
			<td>10K MWCO, 0.1 mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Float Buoys for 0.1mL Slide-A-Lyzer MINI Dialysis Devices</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">69588</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">LSE Digital Dry Bath Heaters</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">1168H25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Thermo Scientific&#160;1300 Series A2 Class II, Type A2 Bio Safety Cabinets</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">13-261-308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Software</td>
			<td style="width:161px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Image Lab Software</td>
			<td style="width:161px;">Bio-Rad</td>
			<td style="width:135px;">1709691</td>
			<td style="width:229px;"></td>
		</tr>
	</tbody>
</table>

co-immunoprecipitation assay using endogenous nuclear proteins from cells cultured under hypoxic conditions

低氧水平 (缺氧) 触发多种适应反应与缺氧诱导因子 1 (HIF-1) 复合体作为主调节器。HIF-1 由 heterodimeric 氧调节α亚基 (HIF-1α) 和组成性表达的β亚基 (HIF-1β) 也称为芳烃受体核转运体 (ARNT), 调节包括血管生成在内的不同过程中所涉及的基因、红细胞和糖酵解。HIF-1 相互作用蛋白的鉴定是了解缺氧信号通路的关键。除了调节 HIF-1α稳定性外, 缺氧还触发了许多转录因子的核易位, 包括 HIF-1α和 ARNT。值得注意的是, 目前用于研究这种蛋白质-蛋白质相互作用 (PPIs) 的方法大多是基于那些通过蛋白质过度表达人为地增加蛋白质水平的系统。蛋白质过度表达往往导致非生理结果的时间和空间文物。在这里, 我们描述了在低氧治疗后使用内源核蛋白的改进的免疫沉淀协议, 并作为概念的证明, 以显示 HIF-1α和 ARNT 之间的相互作用。在本议定书中, 缺氧细胞是在缺氧条件下收获的, Dulbecco 的磷酸盐缓冲盐水 (DPBS) 洗涤缓冲液也在使用前平衡缺氧条件, 以减轻蛋白质降解或蛋白质复合体复氧过程中的离解。此外, 核分数随后提取, 以集中和稳定内源性核蛋白, 并避免可能的假结果往往看到在蛋白质过度表达。该协议可用于证明转录因子与转录联合调节器在缺氧条件下的内源和本机相互作用。

为了评估细胞对缺氧的反应, 研究了缺氧治疗后 HIF-1 复合物的表达水平和亚单位定位。HEK293A 细胞在缺氧条件下培养4小时或保持在 normoxia 作为控制。HIF-1α和 ARNT 蛋白水平检查在整个细胞或核/细胞质提取物的西方印迹。按照预期, 缺氧上调总 HIF-1α水平, 而 ARNT 总细胞裂解物水平没有显著改变 (图 1A)。此外, 缺氧导致 HEK293A 细胞中 HIF-1α和 ARNT 的核积累 (图 1B), 这与以前的报告一致, 尽管在一些经测试的细胞系中未发现 ARNT 的细胞质表达.
接下来, 我们展示了 HIF-1α和 ARNT 在缺氧治疗后的相互作用。核萃取物是从暴露于常氧或缺氧条件下的 HEK293A 细胞中制备的, 用于 4 h. 用 HEK293A 细胞核萃取物进行联合免疫沉淀实验。如图 2所示, HIF-1α与低氧 HEK293A 细胞核萃取物 immunoprecipitated 一起 ARNT。
结合起来, 所描述的协议可以成功地用于诱导细胞缺氧反应, 并进一步确定内在表达 HIF-1α/ARNT 复合体在细胞核内的生理蛋白结合。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57836/57836fig1.jpg"> 图 1:低氧对 HIF-1 组分蛋白表达和亚细胞定位的调节.(A) 对 HEK293A 细胞中总 HIF-1α或 ARNT 表达的应用免疫印迹分析。细胞暴露于缺氧4小时 (h) 或保持在 normoxia (N)。以 anti-HIF-1α、抗 ARNT 和抗甘油 3-磷酸脱氢酶 (GAPDH) 抗体为免疫印迹法, 用 SDS 分页分离出全细胞提取物中的蛋白质。GAPDH 被用作装载控制。(B) 应用免疫印迹分析 HEK293A 细胞亚组分 HIF-1α和 ARNT 表达。HEK293A 细胞在缺氧条件下培养4小时 (h) 或保存在 normoxia (N)。采用 anti-HIF-1α免疫印迹法、抗 ARNT、抗阴、阳 1 (YY1) 和抗 GAPDH 抗体, 对25µg 核或细胞质提取物中蛋白质进行了分析。YY1 和 GAPDH 分别用作核和细胞质馏分的加载控制。<a href="/files/ftp_upload/57836/57836fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57836/57836fig2.jpg"> 图 2:HIF-1α在缺氧条件下与 ARNT 相互作用.HEK293A 细胞暴露于缺氧4小时或保持在 normoxia 作为控制。制备了 HEK293A 细胞的核萃取物, 并用抗 ARNT 抗体进行了 immunoprecipitations。采用 anti-HIF-1α、抗 ARNT、anti-YY1 抗体免疫印迹法, 对核萃取物 (输入) 和 immunoprecipitated 蛋白进行了分析。YY1 被用作输入的加载控制, 而 IgG 作为免疫沉淀实验的负控制。<a href="/files/ftp_upload/57836/57836fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>解决 方案</td>
			<td>组件</td>
			<td>评论</td>
		</tr>
<tr>
<td>透析缓冲液</td>
			<td>20毫米三盐酸 (pH 7.4), 20% 甘油, 100 毫米氯化钾, 0.2 毫米 EDTA, 0.2 毫米 PMSF 和0.5 毫米</td>
			<td>在使用前立即添加 PMSF 和德勤。</td>
		</tr>
<tr>
<td>运行缓冲区</td>
			<td>2.5 毫米三盐酸 (PH 8.3), 19.2 毫米甘氨酸和 0.01% SDS</td>
			<td>混合100毫升10x 三/甘氨酸/SDS 缓冲器与900毫升 ddH2O。</td>
		</tr>
<tr>
<td>传输缓冲区</td>
			<td>2.5 毫米三盐酸 (PH 8.3), 19.2 毫米甘氨酸和20% 甲醇</td>
			<td>混合100毫升10x 三/甘氨酸缓冲器与200毫升甲醇和700毫升 ddH2O。</td>
		</tr>
<tr>
<td>tb 缓冲区</td>
			<td>50毫米三氯 (pH 7.6) 和150毫米氯化钠</td>
			<td>混合100毫升 10x tb 缓冲器与900毫升 ddH2O。</td>
		</tr>
<tr>
<td>缓冲器</td>
			<td>50毫米三氯 (pH 7.6), 150 毫米氯化钠和0.1% 吐温20</td>
			<td>混合100毫升 10x tb 缓冲器与900毫升 ddH2O 和1毫升的吐温20。</td>
		</tr>
<tr>
<td>阻塞缓冲区</td>
			<td>50毫米三氯 (pH 7.6), 150 毫米氯化钠, 0.1% 吐温20和5% 非脂牛奶</td>
			<td>溶解5克的印迹级阻滞剂在100毫升的 tb 吨缓冲。</td>
		</tr>
<tr>
<td>IP 缓冲区</td>
			<td>50毫米三盐酸 (pH 7.4), 180 毫米氯化钠, 20% 甘油, 0.2% NP-40 和1x 蛋白酶抑制剂鸡尾酒</td>
			<td>在使用前立即加入蛋白酶抑制剂鸡尾酒。</td>
		</tr>
</tbody></table>表 1: 解决方案的准备工作。

Watch this Scientific Journal Video about Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions at JoVE.com

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

在这里, 我们描述了一个共同免疫沉淀协议, 研究在缺氧条件下内源核蛋白的蛋白质相互作用。该方法适用于低氧转录因子与转录联合调节剂相互作用的证明。

缺氧条件下培养细胞内源性核蛋白的共免疫沉淀测定

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...

co-immunoprecipitation-assay-using-endogenous-nuclear-proteins-from

Nanyang Technological University

Research

JoVE Journal

Biology

17.1K 次观看.  Nanyang Technological University. 在这里, 我们描述了一个共同免疫沉淀协议, 研究在缺氧条件下内源核蛋白的蛋白质相互作用。该方法适用于低氧转录因子与转录联合调节剂相互作用的证明。

视频: Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

根据流量的血小板粘附和聚集，使用微流控流细胞

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

科研

生物学

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

染色体制备培养细胞

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Anti-Nuclear Antibody Screening Using HEp-2 Cells

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although IIF is an excellent screening test in expert hands, the technical difficulties of processing and reading IIF slides &#8211; such as the labor intensive slide processing, manual reading, the need for experienced, trained technologists and the use of dark room &#8211; make the IIF method difficult to fit in the workflow of modern, automated laboratories.
The first and crucial step towards high quality ANA screening is careful slide processing. This procedure is labor intensive, and requires full understanding of the process, as well as attention to details and experience.
Slide reading is performed by fluorescent microscopy in dark rooms, and is done by trained technologists who are familiar with the various patterns, in the context of cell cycle and the morphology of interphase and dividing cells. Provided that IIF is the first line screening tool for SARD, understanding the steps to correctly perform this technique is critical.
Recently, digital imaging systems have been developed for the automated reading of IIF slides. These systems, such as the NOVA View Automated Fluorescent Microscope, are designed to streamline the routine IIF workflow. NOVA View acquires and stores high resolution digital images of the wells, thereby separating image acquisition from interpretation; images are viewed an interpreted on high resolution computer monitors. It stores images for future reference and supports the operator&#8217;s interpretation by providing fluorescent light intensity data on the images. It also preliminarily categorizes results as positive or negative, and provides pattern recognition for positive samples. In summary, it eliminates the need for darkroom, and automates and streamlines the IIF reading/interpretation workflow. Most importantly, it increases consistency between readers and readings. Moreover, with the use of barcoded slides, transcription errors are eliminated by providing sample traceability and positive patient identification. This results in increased patient data integrity and safety.
The overall goal of this video is to demonstrate the IIF procedure, including slide processing, identification of common IIF patterns, and the introduction of new advancements to simplify and harmonize this technique.

Indirect immunofluorescent (IIF) assays have traditionally been used for the detection of antinuclear antibodies (ANA) in human serum. The presence of these antibodies can aid in the diagnosis of systemic autoimmune rheumatic diseases (SARD). This protocol demonstrates how to effectively perform the IIF technique to accurately detect these autoantibodies.

抗核抗体筛选使用Hep-2细胞

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

内源性的免疫荧光分析及外源着丝粒着丝粒蛋白

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

单探头的应用:根据环境条件质谱成像和单细胞分析

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

无细胞测定中使用<em&gt;非洲爪蟾</em&gt;胚胎提取研究核的大小调节机制。

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.

使用矫形器（40-300 kV）X射线设施进行细胞辐照的测定

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose ...

Field-Based Thermal Physiology Assay: Cold Shock Recovery under Ambient Conditions

Ecological physiology, particularly of ectotherms, is increasingly important in this changing world as it uses measures of species and environmental traits to explore the interactions between organisms and their surroundings to better understand their survival and fitness. Traditional thermal assays are costly in terms of time, money, and equipment and are therefore often limited to small sample sizes and few species. Presented here is a novel protocol that generates detailed data on individual behavior and physiology of large, volant, terrestrial insects, using the example of butterflies. This paper describes the methods of a cold shock recovery assay that can be performed in the field under ambient environmental conditions and does not require costly laboratory equipment. This method has been used to understand the response and recovery strategy to cold shock of tropical butterflies, generating individual level data across entire butterfly communities. These methods can be employed in both remote field settings and classrooms and can be used to generate ecologically relevant physiological data and as a teaching tool.

Here, a low-cost, accessible protocol is described to evaluate cold shock recovery of butterflies under ambient environmental conditions.

基于现场的热生理学测定:环境条件下的冷休克恢复

Ecological physiology, particularly of ectotherms, is increasingly important in this changing world as it uses measures of species and environmental ...