Name: study of protein-protein interactions in autophagy research
Uploaded: 2017-09-09T13:00:00.000+00:00
Duration: 14 min 8 s
Description: Watch this Scientific Journal Video about Study of Protein-protein Interactions in Autophagy Research at JoVE.com

这项工作得到了土耳其科学和技术研究理事会 (研究) 1001 赠款 107T153, 萨班吉大学的支持。赛博和 NMK 的支持下, 研究 BIDEB 2211 奖学金的博士学位研究。

1. 免疫荧光 <ol> <li> 在 DMEM 高糖培养基中维持 HEK293T 人胚肾细胞, 在 DMEM 低血糖培养基中 N2A 小鼠 neuroblast 细胞, 在 5% CO 2 加湿培养箱中, 37 和 #176;用10% 热灭活胎牛 (FBS) 血清、抗生素 (50 U/毫升青霉素、50和 #181、g/毫升链霉素) 和 l-谷氨酰胺 (2 mM) 补充培养基. </li>
	<li> 使用0.25% 胰蛋白酶分离细胞。首先删除细胞培养的培养基, 然后用10毫升无菌磷酸缓冲盐 (PBS) 冲洗细胞, 在37和 #176 中孵育1毫升的胰蛋白酶; C 为5分钟, 添加2毫升 DMEM 培养基, 使胰蛋白酶失活. </li>
	<li> 用移把盘子洗好。移除10和 #181; l 细胞溶液, 并与10和 #181 混合; 我的台盼蓝。使用例计算活单元数. </li>
	<li> HEK293T 和 N2A 都是粘附的细胞系, 但是, HEK293T 细胞很容易从钢板上分离出来。因此, 如果使用 HEK293T 细胞系, 在播种细胞之前, 用无菌过滤的0.01% 聚 l-赖氨酸涂层 coverslides. 
	<ol> <li> 将无菌 coverslides 放置在10厘米的培养皿中。在每个 coverslide 上加入适量的聚 l-赖氨酸溶液。用聚 l-赖氨酸溶液孵育 coverslides 10 分钟. </li>
		<li> 移除聚 l-赖氨酸溶液. 
		注: 聚 l-赖氨酸是可重用的;它是可选的收集和重用它作进一步的实验。由于聚 l-赖氨酸是有毒的, 等待, 直到所有的解决方案上的 coverslides 完全蒸发, 然后恢复聚 l-赖氨酸. </li>
		<li> 用无菌 PBS 清洗 coverslides. </li>
	</ol> </li> <li> 将 1 mL DMEM 添加到12个井板的每个井中。每井放置一个 coverslide. </li>
	<li> 种子2万细胞/井, 在同时摇动板时下落, 以使 coverslides 上的细胞均匀分布。将 CO 2 孵化器中的单元格保持在37和 #176; C 和添加药物, 使总潜伏期不超过48小时, 即 , 3 润治疗, 添加药物在 45 h 后播种. 
	<ol> <li> 用于 RACK1-LC3 相互作用研究, 润 1, 雷帕霉素和饥饿被用作自噬诱导。细胞孵育与润1为 3 h (250 毫微米最后的集中);与雷帕霉素为 16 h (200 毫微米最后的集中);与厄尔和 #39 的平衡盐溶液 (EBSS) 为2小时, 以饿死细胞. 
		注: 以 10% FBS 的 DMEM 为控制条件. </li>
	</ol> </li> <li> 在总潜伏期48小时后, 取出 coverslides 上的介质并用1毫升 PBS 清洗. </li>
	<li> 固定细胞, 孵育细胞与1毫升冰冷无菌过滤4% 甲醛 (粉煤灰) 在 1x PBS (pH 7.4), 在室温下20分钟没有任何躁动. 
	由于粉煤灰对光敏感, 在潜伏期时保持 (重要) 在黑暗中的板块;用铝箔盖板可能有帮助。此外, 粉煤灰是有毒的, 因此, 工作在通风罩, 而固定的细胞. 
	注意: 请记住, 免疫荧光实验中的固定步骤应该优化, 因为特定的抗体可能使用不同的固定剂和条件. </li>
	<li> 在20分钟的粉煤灰孵化后, 首先删除 coverslides 上的煤灰溶液, 然后用1毫升无菌过滤 PBS 洗涤每个样品3次。在这一步, (重要) 一个一个地洗井, 以免让他们干。避免干燥, 因为它会干扰细胞的后续反应, 改变细胞的形态, 并增加非特异性信号. </li>
	<li> 清洗完成后, 将样品保存在1毫升的冰上. </li>
	<li> 准备阻塞解决方案: PBS 与0.1% 牛血清白蛋白 (BSA) 和0.1% 皂甙. </li>
	<li> 用石蜡薄膜覆盖6层板。确保板底在石蜡膜覆盖后光滑。标签6井板. </li>
	<li> 使用镊子, 将 coverslides 从12井板转移到覆盖有石蜡膜的6层板上. </li>
	<li> 在 coverslides 上丢弃 PBS, 并在每个 coverslide 上添加100和 #181; 性。在冰上孵育样品30分钟. </li>
	<li> 准备阻断溶液中的主抗体。在实验之前, 优化抗体稀释, 以确定最佳工作浓度的染色. 
	注: 例如, LC3 抗体在阻断溶液中以1:100 稀释的方式使用. </li>
	<li> 30 分钟孵化后, 丢弃堵塞溶液, 用1毫升 PBS 轻轻冲洗样品一次. </li>
	<li> 在每个 coverslide 中添加100和 #181; 主要抗体溶液将样品在室温下孵育1小时, 放在振动筛上. 
	注意: 与主抗体的孵育时间应优化。根据抗体, 夜间孵化在4和 #176; C 可能是可取的。最好在摇床上轻轻地孵育 (100 轮/分钟), 以便在 coverslides 上均匀地分布抗体. </li>
	<li> 孵育后与主抗体, 单独洗涤样本, 3 次与1毫升 PBS. </li>
	<li> 在阻塞解决方案中准备1:500 次抗体 (可能需要对二次抗体的稀释进行优化)。在这里, 兔 IgG Alexa 568 被用作第二兔抗体. 
	<ol> <li> 将100和 #181 的样品孵育到一个振动筛的室温下的二次抗体溶液 L。由于荧光标记的二级抗体对光敏感, (重要) 在黑暗中保持溶液。在二次抗体孵化期间和之后放置样品在黑暗中. </li>
	</ol> </li> <li> 继二次抗体孵化后, 用 PBS 冲洗盘子3次. 
	注: 基于根据经验, 一些抗体有高的背景信号, 不能消除 PBS 洗。如果处理这种抗体, 在步骤1.21 之后, 在 PBS 中保持样品在4和 #176; C 为 6 h 可能帮助。
	<ol> <li> 如果需要, 用 DNA 结合染料 (如赫斯特) 将细胞染色, 以标记细胞核. </li>
	</ol> </li> <li> 要对多个蛋白质进行着色, 请在第二个蛋白质的步骤1.16 开始应用相同的过程. 
	<ol> <li> 确保在执行带有多个抗体的染色 ( 例如 、使用鼠标和兔抗体) 时, 抗体之间没有物种反应性。
		注: 在这里, RACK1 被染色作为第二个蛋白质使用 anti-RACK1 主要抗体和 anti-mouse IgG Alexa 488 次抗体使用相同浓度上述. </li>
	</ol> </li> <li> 准备安装解决方案: 1x PBS 中的50% 甘油。筛选通过22和 #181; m 过滤器. </li>
	<li> 使用无皮棉擦拭擦除带有乙醇的幻灯片, 并添加10和 #181; 每张幻灯片的安装解决方案。使用镊子, 将 coverslides 放在每个 dropso 上, 与单元格的侧面直接接触安装介质。使用无绒抹布轻轻地丢弃多余的安装解决方案. </li>
	<li> 用透明指甲油密封 coverslides。首先, 通过在4棱上加入滴指甲油来稳定 coverslides, 然后完全密封. </li>
	<li> 在荧光或共聚焦显微镜下尽快分析样品, 因为荧光信号可能在几个小时后漂白. </li>
</ol> p 类 = "jove_title ">> 2。沉淀 <ol> <li> 分离和计数单元格, 如步骤1.2 和1.3 所述. </li>
	<li> 如果使用 HEK293T 单元格, 则在 15 cm 的培养皿中为每个条件种子400万细胞。如果工作与 N2A 细胞, 种子500万细胞在 15 cm 培养皿。在37和 #176 的 CO 2 孵化器中孵育细胞, 直到实验结束. </li>
	<li> 在定义的持续时间内, 使用相关药物或条件对细胞进行治疗, 使细胞的总潜伏期不超过48小时. 
	注: 对于本研究中的沉淀试验, 细胞被治疗 3 h 与润 1 (250 nm), 16 h 与雷帕霉素 (200 nm), 和 2 h 与 EBSS。DMEM 以 10% FBS 作为控件添加到单元格中. </li>
	<li> 在总孵育时间48小时后, 取出介质并采集细胞。由于 HEK293T 细胞很容易从钢板上分离出来, 用冰冷 PBS 冲洗盘子可能就足够了。然而, N2A 细胞更耐脱落;使用单元格刮刀来帮助收割细胞. 
	<ol> <li> 收集离心管中的所有单元格. </li>
	</ol> </li> <li> 在4和 #176 上离心取样, 15 分钟 1.6万 x g; c. 用1毫升 PBS 去除上清液, 用 re-suspending 移. </li>
	<li> 重复离心机步骤, 但在最高离心机速度为4和 #176; C 为15分钟, 丢弃上清液. </li>
	<li> 在开始实验前准备无线电免疫沉淀化验 (帕) 缓冲器. 
	注意: 有3种类型的帕缓冲区;根据被调查的蛋白质的相互作用强度选择 (参见下面的公式)。
	<ol> <li> 如果存在强烈的蛋白质-蛋白质相互作用, 请选择常规的帕缓冲区. </li>
		<li> 如果交互较弱, 请选择轻度帕或较温和的帕. 
		注意: 这些缓冲区的配方包括以下步骤。所有在沉淀测试中使用的缓冲器都应辅以蛋白酶抑制剂。
		<ol> <li> 使用 1% NP-40、150毫米氯化钠、0.5% 钠胆酸和0.1% 十二烷基硫酸钠在 50 mM 的 pH 值8中准备常规帕缓冲. </li>
			<li> 用 1% NP-40、150毫米氯化钠和0.25% 钠胆酸在 50 mm 的 pH 7.5 中制备轻度帕缓冲液. </li>
			<li> 准备较温和的帕缓冲剂, 使用1% 卫三 100, 137 毫米氯化钠, 1% 甘油, 1 毫米钠钒在50毫米, pH 7.4. </li>
		</ol> </li> </ol> </li> <li> 选择适当的帕缓冲区后, 用蛋白酶抑制剂 (帕 +) 补充缓冲。然后, 溶解的细胞与3x 的细胞颗粒的体积在帕 +. </li>
	<li> 涡旋暂停十五年代. 在冰上保持5分钟重复涡和结冰5次, 然后离心样品在 1.6万 x g 为 15 min 在4和 #176; C. </li>
	<li> 将上清液转移到清洁的离心管和离心机上, 去除残留的细胞碎片。把上清液转移到干净的管子上. </li>
	<li> 稀释裂解1:50 的蛋白质, 并准备一个具有空白和标准的96井板。稀释样品. 
	<ol> <li> 将示例与布拉德福德解决方案1:20 混合使用。在黑暗中孵育15分钟. </li>
		<li> 测量 595 nm 波长的光学密度。使用每个样本的吸光度值计算蛋白质浓度. </li>
	</ol> </li> <li> 在捕获单元格之前的前一天, 将蛋白质 A 加一个带有特定于感兴趣蛋白质的抗体的念珠。
	<ol> <li> 剪切200和 #181 的边缘; 使用剪刀并使用25和 #181; 从每个条件的珠浆中 l. </li>
		<li> 一次用1毫升 PBS 洗涤, 一次用1毫升帕缓冲, 一次用1毫升帕 +。在 3300 x g, 4 和 #176 执行洗涤步骤; C 为 1 min. </li>
		<li> 最后一次洗涤后, 添加300和 #181; 帕 + 上的珠子, 并添加1.5 和 #181; g 抗体, 是特定的蛋白质被拉下来. </li>
		<li> 在4和 #176 上过夜, 在一个转子 (20 旋转/分钟) 上的 C 上孵化试管. 
		注: 在这里, ATG5 是 immunoprecipitated 使用特定的 anti-ATG5 抗体. </li>
	</ol> </li> <li> 在一夜的念珠与抗体的孵化后, 一次用 PBS 洗涤珠子, 一次用帕, 一次用帕 + 如步骤2.12.2 所述. </li>
	<li> 在每个条件下的珠子上添加2毫克的蛋白质, 并完成总体积到300和 #181; 帕 + 缓冲/管。在4和 #176 的转子上一夜间孵育试管; C. </li>
	<li> 在孵化后, 如前面所描述的那样洗涤珠子 (步骤 2.12.2). 
	<ol> <li> 在最后一次清洗后, 使用较小的吸管提示来除去所有上清液而不接触珠子. </li>
		<li> 添加10和 #181; L 3x 加载染料: 6% SDS, 30% 甘油, 16% 和 #946;-基和0.1% 溴蓝色在1M 三盐酸 pH 6.8. </li>
	</ol> </li> <li> 使用至少100和 #181 准备输入控件; 每个条件的 g 蛋白裂解。使用水平衡样品体积, 并加入适量的3x 负载染料. </li>
	<li> 在95和 #176 中煮沸样品; C 为10分钟. 
	注: 通常在煮沸样品时, 管子的盖子可以自发地打开, 样品也会丢失。通过用特定的盖帽阻止器来保持瓶盖的闭合来避免这一点. </li>
	<li> 自旋下来的样本, 并加载到 SDS 页凝胶, 以分离的蛋白质, 根据其大小使用标准的协议. 
	注: 在本研究中的 RACK1 和 ATG5, 样品运行12% 聚丙烯酰胺凝胶在 80 v 30 分钟, 然后在 120 v 约90分钟。然而, 对于较小的蛋白质, 如 LC3, 使用15% 聚丙烯酰胺凝胶更适合. </li>
	<li> 在凝胶运行完成后, 使用湿或半干转移方法将蛋白质转移到硝化纤维素或 PVDF 膜上。然后, 在 PBS 的5% 脱脂牛奶中用0.05% 吐温 20 (PBST) 在室温下用1小时的时间来阻挡细胞膜, 以 PBST 3 分钟的冲洗膜, 可为5分. </li>
	<li> 在室温下, 在工作浓度为1小时的基础抗体孵育膜。对于初级抗体稀释, 使用含有红色溶液的 bsa (5% bsa V 分数, 0.02% PBST 中叠氮化钠, ph 值 7.5; 添加酚红色作为 ph 值标记), 以允许重复使用抗体. 
	注: 例如, 在红色溶液中稀释 ATG5 抗体2,000x。
	<ol> <li> 在潜伏期结束时, 用 PBST 冲洗膜3次. </li>
	</ol> </li> <li> 使用在5% 脱脂牛奶溶液中制备的 HRP 共轭二级抗体孵育膜 (这里, 兔 IgG 在 1:10.000)。然后用 PBST 冲洗膜 3x 5 分钟. </li>
	<li> 制作 ECL 解决方案:25 mm 鲁米诺、9 mm 豆酸、3 x 10 -4 % H 2 O 2 在 70 mm 中, 三盐酸 pH 8.8。对每个实验使用新的 H 2 O 2 . 
	注: 在 H 2/su 后开始反应b >> O 2 加法, 它持续约20分钟。
	<ol> <li> 在 ECL 中孵育20分钟的膜和 X 射线胶片。开发和修复他们在一个黑暗的房间. </li>
	</ol> </li> <li> 要检查具有相近或重叠分子量的蛋白质的 co-immunoprecipitation, 可能需要通过在60和 #176 上孵化膜来剥离细胞膜上的抗体; 在剥离缓冲器中为30分钟: (25 毫米的三-HCl pH2和 1% SDS). </li>
	<li> 重复步骤2.20 到2.23 以检查使用不同抗体的 co-immunoprecipitation。例如, 在 ATG5 检测和剥离膜后, RACK1 蛋白在同一膜上使用 anti-RACK1 原抗体 (1:1、000) 和二级抗体 (1:10,000) 进行测试。和 #946;-肌动蛋白用作加载控制;用 IgG 信号作为 immunoprecipitated 样品的加载控制. </li>
</ol>

<ol>
	<li>Oral, O., Akkoc, Y., Bayraktar, O., Gozuacik, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+pathological+significance+of+the+molecular+cross-talk+between+autophagy+and+apoptosis.">Physiological and pathological significance of the molecular cross-talk between autophagy and apoptosis.</a> Histol Histopathol. 31, 479-498 (2016).</li><li>Korkmaz, G., Tekirdag, K. A., Ozturk, D. G., Kosar, A., Sezerman, O. U., Gozuacik, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIR376A+is+a+regulator+of+starvation-induced+autophagy.">MIR376A is a regulator of starvation-induced autophagy.</a> PLoS One. 8, e82556 (2013).</li><li>Itah, Z., 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=Hydrodynamic+cavitation+kills+prostate+cells+and+ablates+benign+prostatic+hyperplasia+tissue.">Hydrodynamic cavitation kills prostate cells and ablates benign prostatic hyperplasia tissue.</a> Exp Biol Med (Maywood). 238, 1242-1250 (2013).</li><li>Gozuacik, D., Kimchi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+as+a+cell+death+and+tumor+suppressor+mechanism).">Autophagy as a cell death and tumor suppressor mechanism).</a> Oncogene. 23, 2891-2906 (2004).</li><li>Erzurumlu, Y., Kose, F. A., Gozen, O., Gozuacik, D., Toth, E. A., Ballar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+IBMPFD-related+P97/VCP+mutation+with+differential+binding+pattern+and+subcellular+localization.">A unique IBMPFD-related P97/VCP mutation with differential binding pattern and subcellular localization.</a> Int J Biochem Cell Biol. 45, 773-782 (2013).</li><li>Kuzuoglu-Ozturk, 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=Autophagy-related+gene,+TdAtg8,+in+wild+emmer+wheat+plays+a+role+in+drought+and+osmotic+stress+response.">Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response.</a> Planta. 236, 1081-1092 (2012).</li><li>Longatti, A., Tooze, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+trafficking+and+autophagosome+formation.">Vesicular trafficking and autophagosome formation.</a> Cell Death Differ. 16, 956-965 (2009).</li><li>Erbil, 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=RACK1+Is+an+Interaction+Partner+of+ATG5+and+a+Novel+Regulator+of+Autophagy.">RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy.</a> J Biol Chem. 291, 16753-16765 (2016).</li><li>Gozuacik, D., Yagci-Acar, H. F., Akkoc, Y., Kosar, A., Dogan-Ekici, A. I., Ekici, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticancer+use+of+nanoparticles+as+nucleic+acid+carriers.">Anticancer use of nanoparticles as nucleic acid carriers.</a> J Biomed Nanotechnol. 10, 1751-1783 (2014).</li><li>Phizicky, E. M., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+interactions:+methods+for+detection+and+analysis.">Protein-protein interactions: methods for detection and analysis.</a> Microbiol Rev. 59, 94-123 (1995).</li><li>Uetz, P., 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+comprehensive+analysis+of+protein-protein+interactions+in+Saccharomyces+cerevisiae.">A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae.</a> Nature. 403, 623-627 (2000).</li></ol>

作者声明他们没有竞争的金融利益。

沉淀和免疫荧光技术是研究蛋白质-蛋白质相互作用的关键。虽然这两种技术是常用的和完善的, 但应考虑在使用这些技术时确定实验质量的几个标准。
首先, 在这些测试中使用的主要抗体应该是特定的蛋白质的兴趣。为了确保这一点, 使用 shRNA knockdowns 或敲除细胞测试的特异性抗体的问题。此外, 使用积极的控制, 或治疗细胞与已知的蛋白质诱导的兴趣。批次的抗体的变种也存...

Macroautophagy (自噬) 是一种细胞应激机制, 其特征是在称为噬囊泡的双膜囊泡中的大体积细胞质、蛋白质和胞器的螯合。通过双膜外层的融合, 噬囊泡及其货物被传递到溶酶体并在其中降解1。自噬发生在所有细胞类型和所有生物体的低基底水平, 执行稳态功能, 如蛋白质降解和器官 (如, 线粒体) 周转。在导致细胞压力的条件下, 如饥饿, 自噬迅速上调并允许细胞维持能量水平和基本代谢1,2,3。
从酵母中克隆出大约30自噬基因, 他们的蛋白质产品在噬过程的各个阶段都扮演了一个角色, 包括囊泡形核、扩张、囊泡融合到晚期 endosome/溶, 以及货物降解4,5. 基因这些基因的大多数已经被确定, 并且在各种生物体中的研究证实了它们的细胞功能的保存情况6。在过去十年的研究表明, 几个自噬相关的蛋白质复合物和蛋白质-蛋白质相互作用存在, 他们控制自噬通路的复杂和控制的方式。交叉路口, 备份, 反馈和前馈机制存在, 他们允许细胞协调自噬与其他相关事件 (如水泡分泌物, 溶生物, 内涵排序和传输7,等)在一个无偏酵母-两个混合屏幕使用自噬蛋白 ATG5 作为诱饵, (ATG5 是一个关键的吞噬蛋白参与 E2-like 共轭系统, 介导 LC3 lipidation 在饥饿诱导自噬), 我们已经确定受体激活c-激酶 1 (RACK1;GNB2L1) 作为一个强大的交互和一个新的自噬成分8。重要的是, 屏幕显示, ATG5-RACK1 相互作用是不可缺少的自噬诱导的经典吞噬诱导 (即, 饥饿和 mTOR 抑制)。
Immunofluorescence-based 方法通常用于监测蛋白质-蛋白质相互作用。这些技术主要是抗体, 有助于可视化交互和确认细胞定位。在这种技术中, 荧光标记共轭抗体是特定的蛋白质的兴趣通常用于特定的染色。每种蛋白质都可以贴上不同荧光染料的抗体。使用蛋白质特异抗体, 在图像被合并时信号的重叠表明在共焦显微镜下蛋白质的共存。该技术适用于细胞甚至组织。免疫荧光技术提供了有关相互作用动力学的线索, 帮助确定蛋白质复合体的大小和分布, 同时跟踪不同条件下细胞形态学的一般变化9。沉淀是另一种常用的抗体技术, 允许分析给定蛋白质之间的相互作用10。利用这一技术, 感兴趣的蛋白质从细胞或组织提取物中分离出来, 使用特定的抗体, 导致蛋白质沉淀在一个复杂的或与感兴趣的蛋白质接触。共沉淀, 其中蛋白质和其 co-interactor 的检测, 不仅揭示了两个蛋白质之间的相互作用, 但可以测量其在不同情况下的相互作用强度11。
本协议详细描述了用于确认和描述 ATG5-RACK1 和 RACK1-LC3 交互的关键技术。重点是免疫荧光和沉淀技术, 重点是在自噬研究的关键步骤和陷阱, 以及故障排除建议。

<table><tbody><tr><td>Trypsin EDTA Solution A</td><td>Biological Industries&amp;nbsp;</td><td>BI03-050-1A</td><td></td></tr><tr><td>PBS</td><td>GE Healthcare</td><td>SH-30256.01</td><td></td></tr><tr><td>DMEM (high glucose)</td><td>Sigma&amp;nbsp;</td><td>5671</td><td></td></tr><tr><td>DMEM (low glucose)</td><td>Sigma</td><td>5546</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Hemocytometer</td><td>Sigma</td><td>Z359629-1EA</td><td></td></tr><tr><td>coverslides</td><td>Jena Bioscience</td><td>CSL-103</td><td></td></tr><tr><td>slides</td><td>Isolab</td><td>I.075.02.005</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma&amp;nbsp;</td><td>P8920</td><td></td></tr><tr><td>Torin</td><td>Tocris</td><td>4247</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>VWRSAD2650</td><td></td></tr><tr><td>EBSS</td><td>Biological Industries&amp;nbsp;</td><td>BI02-010-1A</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>15812-7</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>&amp;nbsp;A4503</td><td></td></tr><tr><td>Saponin</td><td>Sigma</td><td>84510</td><td></td></tr><tr><td>LC3 Antibody</td><td>Sigma</td><td>L7543</td><td></td></tr><tr><td>Anti-Rabbit IgG Alexa Fluor 568</td><td>&amp;nbsp;Invitrogen</td><td>A11011</td><td></td></tr><tr><td>RACK1 Antibody</td><td>Santa Cruz Biotechnology</td><td>&amp;nbsp;sc-17754</td><td></td></tr><tr><td>Anti-Mouse IgG Alexa Fluor 488</td><td>&amp;nbsp;Invitrogen</td><td>A11001</td><td></td></tr><tr><td>NP-40</td><td>Applichem</td><td>A16694.0250</td><td></td></tr><tr><td>Sodium Chloride</td><td>Applichem</td><td>A9242.5000</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td>30970</td><td></td></tr><tr><td>Sodium dodecyl sulphate (SDS)</td><td>Biochemika</td><td>A2572</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>Triton-X&amp;nbsp;</td><td>Applichem</td><td>4975</td><td></td></tr><tr><td>Sodium orthovanadate</td><td>Sigma</td><td>450243</td><td></td></tr><tr><td>ATG5 Antibody</td><td>Sigma</td><td>A0856</td><td></td></tr><tr><td>Glycerol</td><td>Applichem</td><td>A4453</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol&amp;nbsp;</td><td>Applichem</td><td>A1108.0250</td><td></td></tr><tr><td>Bromophenol blue&amp;nbsp;</td><td>Applichem</td><td>A3640.0005</td><td></td></tr><tr><td>Non-Fat milk</td><td>Applichem</td><td>A0830</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P5927</td><td></td></tr><tr><td>Sodium Azide</td><td>Riedel de Haen</td><td>13412</td><td></td></tr><tr><td>Phenol red&amp;nbsp;</td><td>Sigma</td><td>114537-5G</td><td></td></tr><tr><td>anti-rabbit IgG , HRP conjugated</td><td>Jackson Immuno.&amp;nbsp;</td><td>1110305144</td><td></td></tr><tr><td>Luminol</td><td>Fluka</td><td>9253</td><td></td></tr><tr><td>Coumeric Acid</td><td>Sigma</td><td>C9008</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Merck</td><td>K35522500604</td><td></td></tr><tr><td>anti mouse IgG, HRP conjugated</td><td>Jackson Immuno.</td><td>115035003</td><td></td></tr><tr><td>&amp;beta;-Actin Antibody</td><td>Sigma&amp;nbsp;</td><td>A5441</td><td></td></tr><tr><td>Normal rabbit serum</td><td>Santa Cruz Biotechnology</td><td>sc-2027</td><td></td></tr><tr><td>Rapamycin</td><td>Sigma</td><td>&amp;nbsp;R0395</td><td></td></tr><tr><td>Protein A-Agarose Beads</td><td>Santa Cruz Biotechnology</td><td>sc-2001</td><td></td></tr><tr><td>fetal bovine serum&amp;nbsp;</td><td>Biowest</td><td>S1810-500</td><td></td></tr><tr><td>penicillin/streptomycin solution</td><td>Biological Industries&amp;nbsp;</td><td>03-031-1B</td><td></td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>Biological Industries&amp;nbsp;</td><td>BI03-020-1B</td><td></td></tr><tr><td>Bradford Solution</td><td>Sigma</td><td>6916</td><td></td></tr><tr><td>Nitocellulose membrane</td><td>GE Healthcare</td><td>A10083108</td><td></td></tr><tr><td>X-ray Films</td><td>Fujifilm</td><td>47410 19289</td><td></td></tr><tr><td>Protease inhibitor</td><td>Sigma</td><td>P8340</td><td></td></tr></tbody></table>

study of protein-protein interactions in autophagy research

蛋白质-蛋白质相互作用对于理解细胞信号的叶栅和识别新的通路成分和蛋白质动力学是非常重要的。大多数细胞活动需要蛋白质之间的物理相互作用。为了分析和映射这些相互作用, 开发了各种实验技术以及生物信息学工具。自噬是一种细胞回收机制, 它允许细胞处理不同的压力源, 包括营养匮乏, 化学物质和缺氧。为了更好地了解自噬相关的信号事件, 并发现新的因素, 调节蛋白质复合体自噬, 我们进行蛋白质-蛋白质相互作用的屏幕。验证这些筛选结果需要使用免疫荧光和沉淀技术。在这个系统中, 我们发现的特定的自噬相关蛋白-蛋白质相互作用在 Neuro2A (N2A) 和 HEK293T 细胞系中进行了测试。在这个形象化的实验文件中解释了所使用的技术程序的细节。

在图 1中显示了使用此协议获得的共存结果的示例。我们最近的一篇论文中的一个数字显示为8。在这里, 内源性 RACK1 蛋白呈绿色染色, 内源性 LC3 染红。在合并的图片中观察到的黄色点表示绿色和红色信号之间的重叠点。因而黄色点代表这二个蛋白质的部份共存。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55881...

Watch this Scientific Journal Video about Study of Protein-protein Interactions in Autophagy Research at JoVE.com

Study of Protein-protein Interactions in Autophagy Research

本文介绍了两种抗体蛋白-蛋白质相互作用的研究方法: 免疫荧光和沉淀。这些技术适于研究蛋白质之间的物理相互作用, 以发现细胞信号通路的新成分, 以及了解蛋白质动力学。

自噬研究中蛋白质-蛋白质相互作用的研究

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

study-of-protein-protein-interactions-in-autophagy-research

Research

JoVE Journal

Biology

9.1K 次观看.  Sabanci University. 本文介绍了两种抗体蛋白-蛋白质相互作用的研究方法: 免疫荧光和沉淀。这些技术适于研究蛋白质之间的物理相互作用, 以发现细胞信号通路的新成分, 以及了解蛋白质动力学。 

视频: Study of Protein-protein Interactions in Autophagy Research

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

Lipophagy is a selective form of autophagy that involves the degradation of lipid droplets. Dysfunctions in this process are associated with cancer development. However, the precise mechanisms are not yet fully understood. This protocol describes quantitative imaging approaches to better understand the interplay between autophagy, lipid metabolism, and cancer progression.

探索通过脂肪吞噬对脂滴分解代谢的调节

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This ...

科研

癌症研究

<meta charset="utf8"></head><body>评估溶酶体融合受损自噬中 LC3-II 阳性 EV 释放

Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles

(Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf cytoplasmic contents, subsequently fusing with lysosomes for degradation. Beyond the canonical role, autophagy-related genes also modulate a secretory pathway involving the release of inflammatory molecules, tissue repair factors, and extracellular vesicles (EVs). Notably, the process of disseminating pathological proteins between cells, particularly in neurodegenerative diseases affecting the brain and spinal cord, underscores the significance of understanding this phenomenon. Recent research suggests that the transactive response DNA-binding protein 43 kDa (TDP-43), a key player in amyotrophic lateral sclerosis and frontotemporal lobar degeneration, is released in an autophagy-dependent manner via EVs enriched with the autophagosome marker microtubule-associated proteins 1A/1B light chain 3B-II (LC3-II), especially when autophagosome-lysosome fusion is inhibited.
To elucidate the mechanism underlying the formation and release of LC3-II-positive EVs, it is imperative to establish an accessible and reproducible method for evaluating both intracellular and extracellular LC3-II-positive vesicles. This study presents a detailed protocol for assessing LC3-II levels via immunoblotting in cellular and EV fractions obtained through differential centrifugation. Bafilomycin A1 (Baf), an inhibitor of autophagosome-lysosome fusion, serves as a positive control to enhance the levels of intracellular and extracellular LC3-II-positive vesicles. Tumor susceptibility gene 101 (TSG101) is used as a marker for multivesicular bodies. Applying this protocol, it is demonstrated that siRNA-mediated knockdown of syntaxin-6 (STX6), a genetic risk factor for sporadic Creutzfeldt-Jakob disease, augments LC3-II levels in the EV fraction of cells treated with Baf while showing no significant effect on TSG101 levels. These findings suggest that STX6 may negatively regulate the extracellular release of LC3-II via EVs, particularly under conditions where autophagosome-lysosome fusion is impaired. Combined with established methods for evaluating autophagy, this protocol provides valuable insights into the role of specific molecules in the formation and release of LC3-II-positive EVs.

<meta charset="utf8"></head><body>通过细胞外囊泡释放 LC3-II 与细胞内 LC3 阳性囊泡积累的关系的评价

Here, we present the methodology for concisely assessing autophagosome marker LC3-II levels in extracellular vesicles (EVs) by immunoblotting. Analysis for LC3-II levels in EVs, autolysosome formation, and omegasome formation suggests the new role of STX6 in the release of LC3-II-positive EVs when autophagosome-lysosome fusion is inhibited.

通过细胞外囊泡释放 LC3-II 与细胞内 LC3 阳性囊泡积累的关系的评价

(Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf ...

生物化学

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

生物学

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

活细胞成像早期自噬活动：Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

小鼠中自噬激活有氧运动

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

通过使用多光谱成像流式细胞术测量LC3，p62和LAMP1共定位评估自噬通量

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

乳酸脱氢酶螯合测定--一种简便、可靠的测定哺乳动物细胞中自噬性螯合活性的方法

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

分析黑 腹果蝇 幼虫脂肪体中饥饿诱导的自噬

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

自噬研究中蛋白质-蛋白质相互作用的研究

摘要

探索更多视频

此视频中的章节

探索通过脂肪吞噬对脂滴分解代谢的调节

通过细胞外囊泡释放 LC3-II 与细胞内 LC3 阳性囊泡积累的关系的评价

通过细胞外囊泡释放 LC3-II 与细胞内 LC3 阳性囊泡积累的关系的评价

通过细胞外囊泡释放 LC3-II 与细胞内 LC3 阳性囊泡积累的关系的评价

采用先进的3D荧光显微镜定量分析自噬

活细胞成像早期自噬活动：Omegasomes与超越

小鼠中自噬激活有氧运动

通过使用多光谱成像流式细胞术测量LC3，p62和LAMP1共定位评估自噬通量

乳酸脱氢酶螯合测定--一种简便、可靠的测定哺乳动物细胞中自噬性螯合活性的方法

分析黑腹果蝇幼虫脂肪体中饥饿诱导的自噬

自噬研究中蛋白质-蛋白质相互作用的研究

摘要

探索更多视频

此视频中的章节

探索通过脂肪吞噬对脂滴分解代谢的调节

通过细胞外囊泡释放 LC3-II 与细胞内 LC3 阳性囊泡积累的关系的评价

通过细胞外囊泡释放 LC3-II 与细胞内 LC3 阳性囊泡积累的关系的评价

通过细胞外囊泡释放 LC3-II 与细胞内 LC3 阳性囊泡积累的关系的评价

采用先进的3D荧光显微镜定量分析自噬

活细胞成像早期自噬活动：Omegasomes与超越

小鼠中自噬激活有氧运动

通过使用多光谱成像流式细胞术测量LC3，p62和LAMP1共定位评估自噬通量

乳酸脱氢酶螯合测定--一种简便、可靠的测定哺乳动物细胞中自噬性螯合活性的方法

分析黑 腹果蝇 幼虫脂肪体中饥饿诱导的自噬

分析黑腹果蝇幼虫脂肪体中饥饿诱导的自噬