The authors have no acknowledgements.

1.CNTFRα裂解液的蛋白质印迹<ol><li>表达全长sorLA和Myc标签CNTFRα在人胚肾构建使用的pcDNA3.1 /沸石293（HEK293）细胞（ - ）和的pcDNA3.1 / HYG（ - ），分别。转染HEK293细胞与根据manufacturer&#39;s协议使用市售的转染试剂的构建体。选择在介质中稳定的克隆含有150微克/毫升博来霉素和/或500微克/毫升潮霉素金13。 </li><li> HEK293的种子相等数目的转染表达任CNTFRα-Myc的或共表达在两个4孔板CNTFRα-Myc的/ sorLA 如图 1培养修饰Eagle&#39;s培养基（DMEM）的Dulbecco的细胞在补充有10％胎牛血清（FBS），100U / mL青霉素，和在37℃的5％二氧化碳培养箱100微克/毫升链霉素（完全培养基）。 </li><li>等到细胞是50％ - 80％汇合。 </li><li>取下介质如在图1所示CLF-1:井，用Dulbecco磷酸盐缓冲盐水（DPBS）洗涤一次，并用或不用（对照）为10nM CLC添加新鲜完整平台。孵育所述细胞在37℃。 </li><li>孵化的0和5小时后，恢复介质，并将其存储在1.5 mL管4℃。然后添加补充了蛋白酶抑制剂混合物至每孔（1分钟，4℃）1％的Triton X-100的裂解缓冲液（20mM的Tris-HCl，10mM EDTA的，pH值8.0）。随后，将细胞裂解物转移到1.5毫升试管（4℃）。 </li><li>旋转含有中等和细胞裂解物（3000 xg离心，3分钟，4℃）的管和上清液转移至1.5 mL管（4℃）。弃沉淀。转移5μL的各细胞溶解产物的上清液至新的1.5毫升管中 - 使用它们后来测量的蛋白质含量（步骤1.7）。随即，非还原性LDS样品缓冲液添加到所有上清，涡旋样本。 </li><li>使用5微升等分来衡量PROT在细胞裂解物上清液EIN内容使用根据制造商的协议市售蛋白质测定。 </li><li>在95℃5分钟，加热所述样品。 </li><li>受到蛋白质从每个样品的等量还原SDS-PAGE 13和Western印迹13和可视sorLA，β肌动蛋白，和CNTFRα-Myc的含量在培养基，并在细胞裂解物用兔抗sorLA 16（5微克/毫升），小鼠抗β肌动蛋白（0.4微克/毫升）和小鼠抗Myc（1微克/毫升）的抗体和合适的HRP-连接的第二抗体（1:2000）。 </li><li>根据制造商的协议通过密度量化频带。 </li></ol> 2.免疫细胞化学与溶酶体酶抑制剂组合<ol><li>种子HEK293细胞与CNTFRα-Myc的/ sorLA稳定转染在一个4孔板盖滑动， 如图2。培养的细胞中完整的介质如上述。 </li><li>等到细胞融合是20 - 40％。 </li><li>从孔中除去培养基，并添加有或没有50微克/毫升亮肽素和胃酶抑素A（亮氨酸/ PEP）（抑制溶酶体水解酶）的细胞的完整平台， 如图2所示。孵育所述细胞在37℃。该介质必须每6小时温育的下列24小时期间更换。 </li><li>温育24小时后，再次更换介质（有或无亮氨酸/ PEP）和此时添加10nM的CLC:CLF-1。孵育所述细胞在37℃5小时。 </li><li>在PBS洗涤细胞一次，并修复它们在4％多聚甲醛，pH值7在室温下15分钟。 注意:多聚甲醛是高毒，避免与皮肤，眼睛和粘膜接触。手套和防护眼镜应佩戴和通风橱内的解决方案。 </li><li>在PBS中洗涤细胞一次，并透化细胞在室温中皂苷5分钟在PBS中稀释（0.5％皂苷）。 </li><li>稀释的山羊抗 - CNTFRα（1微克/毫升）（特别是不是小鼠抗Myc用于Western印迹）和小鼠抗LAMP-1（15微克/毫升）的抗体在0.5％皂苷，用在RT下2小时的抗体孵育的细胞。 </li><li>洗涤细胞4×（5分钟）在0.5％的皂苷和用驴抗山羊的Alexa 488-和驴抗小鼠Alexa的在RT 2小时568缀合的二抗（6微克/毫升）孵育细胞。 </li><li>在0.5％皂苷洗细胞4×（5分钟），在去离子水（1分钟）两次，并安装在显微镜载片上使用安装介质盖滑动。 </li><li>分析使用具有63X的C-复消色差透镜水浸物镜，NA 1.2激光扫描共焦显微镜的细胞共聚焦显微镜的抗体染色。 </li></ol>在pSTAT3的水平3.免疫印迹检测<ol><li>种子HEK293-sorLA细胞（其表达CNTFRα的内源性水平）的一个4孔板如图3。培养的细胞中完整的介质如上述。 </li><li>等到了细胞是50 - 80％汇合。 </li><li>从孔中除去培养基，用PBS洗一次，以及有或没有10nM的CLC添加新鲜全培养基:CLF-1的细胞作为在图3中表示。孵育所述细胞在37℃5小时。 </li><li>除去培养基，在未补充的DMEM洗涤细胞两次（空白介质;无FBS和抗生素），然后继续培养的细胞在空白介质90分钟（37℃）。 </li><li>再次，去除培养基并用或不用5nM的CLC添加新鲜空白介质:CLF-1（以引发STAT3磷酸化）， 如图3所示。在37℃孵育15分钟。 </li><li>迅速（1分钟，4℃）加入补充了蛋白酶抑制剂混合物和磷酸酶抑制剂的1％的Triton X-100的裂解缓冲液（20mM的Tris-HCl，10mM EDTA的，pH值8.0）除去培养基，裂解细胞鸡尾酒，每孔。随后，将细胞裂解物转移到1.5毫升试管（4℃）。 </li><li>将5μLØf分别上清液至新的1.5毫升管中 - 使用这些后来测量的蛋白质含量（步骤3.8）。随即，非还原性LDS样品缓冲液添加到所有上清，涡旋样本。 </li><li>测量使用根据制造商的协议市售蛋白质测定的5微升等分试样的蛋白质含量。 </li><li>超声处理在RT每个样品直到它们不再粘性（约1分钟），随后在95℃加热它们5分钟。 </li><li>从每个样品进行SDS-PAGE 13和Western印迹13经受蛋白的等量和使用兔抗pSTAT3的（1:2000）形象化pSTAT3的在细胞裂解物的含量和总STAT3和小鼠抗STAT3（1:1000 ）抗体和相应的HRP连接的小鼠和兔第二抗体（1:2000）。 </li><li>根据制造商的协议通过密度量化频带。 </li></ol>

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这里所描述的协议可以专门用于演示sorLA介导CLC:CLF-1依赖性下调和溶酶体CNTFRα靶向，以及伴随的减弱响应于CLC:CLF-1的刺激。 在HEK293细胞系非常适合于这个协议中，因为它们具有CNTFRα和sorLA只有轻微的内源性表达，很容易就可以转染，表达的gp130 /LIFRβ，并具有大的胞体，其是适合进行免疫细胞化学。然而，在理论上具有类似特性的细胞系应正常工作。 该协议有两个关键步骤。第一个问题步骤2.3，其中列伊/ PEP介质必须更换大约每6小时24小时。如果不这样做，可能会导致积极的溶酶体酶，减少或蛋白质的溶酶体中没有检测到积累。第二个关键步骤（3.4）的关注与CLC在预孵化洗:CLF-1。这是IMportant以除去任何未结合的配体，并且以使细胞的时间来恢复（避免持续刺激）在未补充的DMEM（空白培养基）。这将确保以后的再刺激与CLC中pSTAT3的低背景水平:CLF-1（步骤3.5）。 值得注意的是， 图6表明细胞反应（pSTAT3的）与CNTFRα的sorLA介导的下调平行减小。但是，应当强调的是，虽然减少反应是按照（并且在预期）的减少的CNTFRα池的，它并不能证明低CNTFRα表达单独占降低的细胞应答。因此，变化 - 从预刺激或转染得到的 - 中的任何信号通路上游pSTAT3的的功能元件的可能的改变的响应作出了贡献。 该协议的基本概念并不限于此particuLAR受体系统。 （ 即另一个细胞系，其他抗体，其他配体， 等 ）通过修改协议可以被用来确定其他受体的配体诱导的下调，以及-无论sorLA&#39;s参与。然而，应该指出的是，通过Western印迹受体下调的分析主要是适合的受体， 例如 ，GPI锚定受体，它表现出缓慢周转和在未刺激的细胞中低度的新合成的。因此，在受体表示的新合成高水平，配体诱导的下调可用于通过新的受体合成补偿。在这些情况下，该受体池的下调可以改为通过代谢标记和脉冲追踪实验装置如在21中所述测定。可替代地，不与受体干扰碘化抗体（优选的Fc片段）结合可用于"标记"的招待的胞外域或者，与预期的下调然后可以通过细胞沉淀和介质的放射性计数测定。 为物流原因我们转染我们的带CNTFRα细胞构建携带一个Myc-标签，并在本协议被用于受体的Western印迹检测的小鼠抗Myc抗体。与此相反，使用山羊抗CNTFRα抗体进行免疫细胞化学和双标记为LAMP-1是由小鼠抗体检测 - 显然使用两个初级小鼠抗体会导致与第二抗体的非特异性（重叠）染色。注意，由于山羊抗CNTFRα也适用于蛋白质印迹（未示出），该协议可以很容易地进行修改和应用到与未标记CNTFRα转染的细胞。 最后，最近已表明，还内吞受体分拣蛋白结合分类号:CLF-1以高亲和力22。因此，可以想到的是分拣蛋白，像sorLA，互连方介绍:通过CLF-1CNTFRα并能介导的内吞作用和溶酶体目标CNTFRα为好。使用分拣蛋白代替sorLA，本协议可用于澄清这个问题。

在CLC和CLF-1构成异源二聚体细胞因子CLC的两个亚基:CLF-1 1-3用于蜂窝信号感应，CLC:CLF-1的第一目标的CNTFRα，其初级和GPI锚定受体，以形成膜结合三聚体复合物。在CLC:CLF-1:CNTFRα复杂随后招募的gp130 /LIFRβ介导的跨膜通过JAK激酶/信号传感器和转录3（JAK / STAT3）途径4的信号活化剂。小鼠中的任何三个亚基的缺陷显示一个和相同的表型和出生后24小时内死亡，由于面部神经元和乳不足5-8的数量减少。在人类发现同样强调了三个亚基的功能一致性。因此，人类突变（纯合子或化合物）CLC的引起功能障碍:CLF-1或CNTFRα，所有结果在所谓的冷诱导的出汗/ Crisponi综合征，其特征在于症状例如impai一个条件红乳和吞咽，各种畸形特征，温度峰值，和似是而非，灌注低温出汗9-11。 在体外，CLC和CNTFRα的结合是必要的和足够用于与gp130的/LIFRβ相互作用和在单元12的信号的诱导。 CLF-1的另一方面中的作用尚不清楚。它不直接参与的信号，早已被看作是主要用来方便方介绍1细胞分泌的附录。然而，最近的研究结果显示，CLF-1有更多和更重要的功能都牵涉信令和CLC和CNTFRα成交。因此，看来CLF-1含有3个独立的结合位点:一个用于它的公知的结合CLC;一个介导直接结合到CNTFRα;和第三（高亲和力）的网站与内吞受体sorLA相互作用。作为CLC和CLF-1似乎都＆＃160;具有比CLC相当低亲和力靶向CNTFRα:CLF-1复合物，可以想到的是CLF-1（通过其CNTFRα结合位点）促进CLC和CNTFRα的统一，并由此促进信号13。 CLF-1与sorLA互动，本演示的主要问题，扮演着一个完全不同的角色。 SorLA是五个类型1受体构成Vps10p域受体家族14中的一个。它是在各种组织中，但特别是在脑和神经组织15表达。类似于其他家庭成员sorLA携带N末端配体结合Vps10p域，但除此之外，还包括其他的域类型，包括在所述低密度脂蛋白受体家族15成员发现配体结合的元件。其胞质尾与几个接头蛋白， 如接头蛋白-1与-2和sorLA交互传递效率endocytos是以及结合蛋白16-18的细胞内分选和运输。该Vps10p域包括大十刃β螺旋桨19,20，结合一系列不相关的配体，包括CLF-1（但值得注意的是，并非CLC）13。在CLF-1的结合位点是可访问的，即使以后形成复合物与CLC和CNTFRα这意味着sorLA不仅结合游离CLF-1，但也分类号:CLF-1和三聚体复合物CLC:CLF-1:CNTFRα13。 SorLA传达CLF-1和CLC的快速内吞:CLF-1，但这些是可溶性蛋白而CNTFRα被固定到由一个GPI锚的表膜。的问题因此，如果结合CLC的:CLF-1:CNTFRα允许sorLA内化整复，从而改变CNTFRα的面膜表达（和随后的蜂窝易感性CLC:CLF-1信号诱导）和/或CNTFRα的营业额。 在本描述的实验报告旨在阐明以下几个问题: 是否sorLA调解中图分类号:表面膜CNTFRα的CLF-1依赖性下调？为了阐明这一点，它最初试图确定CNTFRα的收入（在不存在和sorLA的存在下）通过的代谢标记和脉冲追踪实验装置如21所述。然而，试图用CNTFRα[S 35]半胱氨酸的混合物，[S 35]甲硫氨酸显示出放射性掺入差标记。这提出了一个非常低的程度的新的合成，它在所有的可能性将是无法弥补的受体的突然和显著损失。要确定是否在通过CLC互连CNTFRα下调:CLF-1 sorLA，有人因此决定之前和暴露于CLC后简单地测量（使用免疫印迹）CNTFRα总细胞池:CLF-1 - 并比较结果在细胞转染或不与染sorLA。 是否sorLA目标CNTFRα到溶酶体？要回答这个问题，CNTFRα的定位 - 通过其CLC内在:CLF-1介导结合sorLA - 利用免疫细胞化学进行了检查，并针对CNTFRα晚期内体/溶酶体标记溶酶体相关的膜蛋白1抗体和标记（LAMP-1）。该实验是在处理过的以及未经处理的同溶酶体酶的抑制剂的细胞进行。当时的想法是，当然，如果CNTFRα在溶酶体降解，用酶抑制剂处理的细胞会提出CNTFRα染色的LAMP-1阳性囊泡积累，而未经处理的细胞会显示CNTFRα很少或根本没有染色。 是否CNTFRα下调降低方介绍细胞反应:CLF-1的刺激？三聚体复合物通过使用JAK / STAT3所述的gp130 /LIFRβ异二聚体由CLC，CLF-1和CNTFRα信号途径。因此，细胞的能力，以应对分类号:在CNTFRα下调CLF-1信号通过Western印迹检测和在sorLA转染的磷酸-STAT3（pSTAT3的）/总STAT3水平的定量探讨。

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			<td>Zeocin</td>
			<td>InvivoGen</td>
			<td>ant-zn-1</td>
			<td></td>
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		<tr>
			<td>Hygromycin B Gold</td>
			<td>InvivoGen</td>
			<td>ant-hg-1</td>
			<td></td>
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			<td>FuGENE 6 Transfection Reagent&#160;</td>
			<td>Roche</td>
			<td>11 815 091 001</td>
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			<td>4-well plates (Nunclon Delta Surface)</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
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			<td>Safe-Lock tubes</td>
			<td>Eppendorf</td>
			<td>0030 120.086</td>
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			<td>Dulbecco Modified Eagle&#180;s Medium</td>
			<td>Lonza</td>
			<td>BE12-604F/U1</td>
			<td></td>
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			<td>Fetal bovine serum</td>
			<td>Thermo Scientific</td>
			<td>10270106</td>
			<td></td>
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			<td>Penicillin-Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140122</td>
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			<td>HyClone</td>
			<td>SH30028.02</td>
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			<td>CLC:CLF-1</td>
			<td>R&#38;D Systems</td>
			<td>1151-CL/CF</td>
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			<td>Mouse anti-LAMP-1&#160;</td>
			<td>DSHP</td>
			<td>H4A3</td>
			<td></td>
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		<tr>
			<td>Rabbit anti-sorLA</td>
			<td></td>
			<td></td>
			<td>Reference 16</td>
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			<td>Mouse anti-b-actin&#160;</td>
			<td>Sigma</td>
			<td>A2228</td>
			<td></td>
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			<td>Mouse anti-Myc</td>
			<td>Genscript</td>
			<td>A00704</td>
			<td></td>
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			<td>Goat anti-CNTFRa&#160;</td>
			<td>R&#38;D Systems</td>
			<td>AF-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-pSTAT3&#160;</td>
			<td>Cell signaling Technology</td>
			<td>9145s</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-STAT3&#160;</td>
			<td>Cell signaling Technology</td>
			<td>9139s</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse HRP-linked antibody</td>
			<td>Cell signaling Technology</td>
			<td>7076s</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit HRP-linked antibody</td>
			<td>Cell signaling Technology</td>
			<td>7074s</td>
			<td></td>
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			<td>Anti-goat HRP-linked antibody</td>
			<td>Dako</td>
			<td>P0160</td>
			<td></td>
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			<td>Donkey anti-goat secondary antibody, Alexa Fluor 488 conjugate</td>
			<td>Invitrogen</td>
			<td>A11055</td>
			<td></td>
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			<td>Donkey anti-mouse secondary antibody, Alexa Fluor 568 conjugate</td>
			<td>Invitrogen</td>
			<td>A10037</td>
			<td></td>
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		<tr>
			<td>Leupeptin</td>
			<td>Sigma</td>
			<td>L8511-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepstatin A&#160;</td>
			<td>Sigma</td>
			<td>P4265-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>AppliChem</td>
			<td>A1388</td>
			<td></td>
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		<tr>
			<td>Tris-HCl</td>
			<td>Merck</td>
			<td>1,082,191,000</td>
			<td></td>
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		<tr>
			<td>EDTA:Titriplex III</td>
			<td>Merck</td>
			<td>1,084,180,250</td>
			<td></td>
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		<tr>
			<td>cOmplete mini EDTA-free</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
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		<tr>
			<td>PhosSTOP</td>
			<td>Roche</td>
			<td>04 906 837 001</td>
			<td></td>
		</tr>
		<tr>
			<td>LDS sample Buffer (4X)&#160;</td>
			<td>Novex</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay</td>
			<td>Bio-Rad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>VWR</td>
			<td>97,135,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>S7900-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Media</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
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sorla and clc:clf-1-dependent downregulation of cntfr&#945; as demonstrated by western blotting, inhibition of lysosomal enzymes, and immunocytochemistry

异二聚体细胞因子心脏营养素状细胞因子:细胞因子样因子1（CLC:CLF-1）靶向糖基（GPI）锚定CNTFRα以形成随后募集糖蛋白130 /白血病抑制因子受体β三聚体复合物（gp130的/ LIFRβ），用于信令。无论CLC和CNTFRα所必需的信号，但到目前为止CLF-1只被称为CLC分泌假定推动者。但是，最近已表明，CLF-1包含三个结合位点:一个用于CLC;一个用于CNTFRα（可以促进三聚体复合物的装配）;和一个用于内吞受体sorLA。后者的网站为高亲和性CLF-1，CLC的结合:CLF-1，以及三聚体（CLC:CLF-1:CNTFRα）复杂sorLA，并在sorLA表达细胞的可溶性配体CLF-1和CLC :CLF-1迅速吸收并内化。在细胞共表达CNTFRα和sorLA，CNTFRα第一结合分类号:CLF-1上，形成膜相关的三聚体复合物，但它也可以通过在CLF-1的自由sorLA结合位点连接到sorLA。其结果，CNTFRα，具有用于在其自己的内吞没有容量，沿着拽和由CLC的sorLA介导的细胞内吞作用内在化:CLF-1。 本协议描述用来演示i）本sorLA介和CLC实验过程:表面膜CNTFRα表达CLF-1相关的下调; II）sorLA介导的内吞作用和溶酶体CNTFRα的目标;以及iii）CLC降低的细胞反应:在CNTFRα的sorLA介导的下调CLF-1刺激。

图1显示了细胞接种和CLC的示意图:在协议CLF-1孵育。 图2示出了细胞接种和亮氨酸/ PEP和CLC的示意图:CLF-1孵育的协议。 图3显示了细胞接种和CLC的示意图:在协议。 图4 CLF-1温育和刺激显示了sorLA介导分类号:CNTFRα的CLF-1依赖性下调。 图5显示了sorLA介导的内吞作用和溶酶体CNTFRα的定位。 图6显示了CLC低响应:CNTFRα下调后CLF-1的刺激。 需要注意的是乐队标志着CNTFRα-Myc的有在零时间相近的密度，而CNTFRα-Myc的下调是由sorLA转较弱的波段AFTE证明R 5小时（ 图4）。 图5显示了CNTFRα-Myc的在亮氨酸/ PEP处理的细胞的LAMP-1阳性囊泡已经积累。与此相反，没有CNTFRα-Myc的积累被认为是在不进行亮氨酸/打气治疗细胞。这表明CNTFRα-Myc的分拣至溶酶体和降解。 CLF-1:如在图6中可以看出，pSTAT3的水平是显著在预刺激的细胞相比，在细胞水平尚未暴露于CLC减少。这是根据在预刺激的细胞CNTFRα的观察sorLA介导的下调（ 图4）。 <img alt="图1" src="/files/ftp_upload/55019/55019fig1.jpg" /> 图1:两个4- CLF-1孵育的协议1.种子的HEK293-CNTFRα-Myc和HEK293-CNTFRα-Myc的/ sorLA细胞:细胞接种和CLC的示意图孔板（0和5小时），并孵育细胞CLC:CLF-1所示。 <a href="http://ecsource.jove.com/files/ftp_upload/55019/55019fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55019/55019fig2.jpg" /> 图2:细胞接种和亮氨酸/ PEP和的示意图CLC:CLF-1孵育的协议2.种子HEK293-CNTFRα-Myc的/ sorLA细胞在4孔板盖玻片孵育细胞有或无亮氨酸/打气和CLC:指示CLF-1。 <a href="http://ecsource.jove.com/files/ftp_upload/55019/55019fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55019/55019fig3.jpg" /> 图3: </s仲&gt; 细胞接种和CLC的示意图:在一个4孔板在协议3种HEK293-sorLA细胞CLF孵化和刺激和培养有或无CLC细胞:（预EXP）CLF-1下调CNTFRα接着CLC:CLF-1刺激（STIM）发起的STAT3磷酸化指示。 <a href="http://ecsource.jove.com/files/ftp_upload/55019/55019fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55019/55019fig4.jpg" /> 图4: 结果显示，SorLA介导图分类号:CNTFRα的CLF-1相关的下调。 （A）的HEK293-CNTFRα-Myc和（B）的HEK293-CNTFRα-Myc的/ sorLA细胞在不存在10nM的CLC的（白色柱）或存在（灰色柱）温育:CLF-1。 0后5小时，孵育停止和CNTFRα-Myc在介质（M）的含量，并在细胞裂解物（L）通过Western印迹检测并通过光密度定量。上部面板显示来自代表性实验Western印迹结果，下图显示CNTFRα-Myc的所检测的水平在细胞裂解物中发现。水平显示相对于0小时CNTFRα-Myc的水平，单，双转。每一列表示平均值±SEM（n = 3时）。 p值用t检验计算。原来的数字，13后转载。 <a href="http://ecsource.jove.com/files/ftp_upload/55019/55019fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55019/55019fig5.jpg" /> 图5:结果显示，SorLA介导的内吞作用和溶酶体CNTFRα定位。 HEK293-CNTFRα-Myc的/ sorLA细胞用或不用亮氨酸/ PEP处理，用10nM CLC孵育:CLF-1 5小时，固定，并最终染色使用抗CNTFRα和抗LAMP-1抗体作为在协议描述2.比例尺:5微米。原来的数字，13后转载。 <a href="http://ecsource.jove.com/files/ftp_upload/55019/55019fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55019/55019fig6.jpg" /> 图6:CNTFRα 的SorLA介导的下调是伴随着方介绍降低的细胞反应:CLF-1的刺激。 HEK293-sorLA细胞预温育在不存在或10nM的CLC的存在:（预实验）CLF-1 5小时，饿死在布劳旱90分钟ķ介质，并用5nM的CLC刺激:（STIM）CLF-1 15分钟。 （ 左侧 ）列显示pSTAT3的在细胞中的相对水平。每一列表示平均值±SEM（n = 3时）相对于所述pSTAT3的水平在没有CLC的预培养细胞:CLF-1，但用CLC刺激:CLF-1。采用t检验计算p值。 （ 右 ）的Western印迹显示了pSTAT3的和STAT3在一个代表性实验中获得的响应。原来的数字，13后转载。 <a href="http://ecsource.jove.com/files/ftp_upload/55019/55019fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about SorLA and CLC:CLF-1-dependent Downregulation of CNTFRÃŽÂ± as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry at JoV

SorLA and CLC:CLF-1-dependent Downregulation of CNTFR&#945; as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

本协议描述印迹和免疫细胞化学以抑制溶酶体酶的组合如何西方可以用来演示睫状神经营养因子受体α（CNTFRα），它是由细胞因子样因子1（CLF-1之间的相互作用输送的下调）和内吞受体sorLA。

SorLA和CLC:这表现在免疫印迹，溶酶体酶的抑制，和免疫组化CNTFRα的CLF-1依赖性下调

The heterodimeric cytokine Cardiotrophin-like Cytokine:Cytokine-like Factor-1 (CLC:CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored ...

sorla-clcclf-1-dependent-downregulation-cntfr-as-demonstrated-western

Research

JoVE Journal

Biochemistry

7.2K 次观看.  Aarhus University. 本协议描述印迹和免疫细胞化学以抑制溶酶体酶的组合如何西方可以用来演示睫状神经营养因子受体α（CNTFRα），它是由细胞因子样因子1（CLF-1之间的相互作用输送的下调）和内吞受体sorLA。 

视频: SorLA and CLC:CLF-1-dependent Downregulation of CNTFRα as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a &#39;splat assay&#39;, which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called &#39;ice-affinity purification&#39;. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification.

植物冰结合蛋白通过冰再结晶抑制和隔离的评估使用冰亲和力纯化鉴定

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein film electrochemistry, in which a protein is immobilized on an electrode surface such that the electrode replaces physiological electron donors or acceptors, has provided functional insight into the redox reactions of a range of different proteins. Full chemical understanding requires electrochemical control to be combined with other techniques that can add additional structural and mechanistic insight. Here we demonstrate a technique, protein film infrared electrochemistry, which combines protein film electrochemistry with infrared spectroscopic sampling of redox proteins. The technique uses a multiple-reflection attenuated total reflectance geometry to probe a redox protein immobilized on a high surface area carbon black electrode. Incorporation of this electrode into a flow cell allows solution pH or solute concentrations to be changed during measurements. This is particularly powerful in addressing redox enzymes, where rapid catalytic turnover can be sustained and controlled at the electrode allowing spectroscopic observation of long-lived intermediate species in the catalytic mechanism. We demonstrate the technique with experiments on E. coli hydrogenase 1 under turnover (H2 oxidation) and non-turnover conditions.

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Here, we describe a technique, protein film infrared electrochemistry, which allows immobilized redox proteins to be studied spectroscopically under direct electrochemical control at a carbon electrode. Infrared spectra of a single protein sample can be recorded at a range of applied potentials and under a variety of solution conditions.

用 [NiFe] 氢对 H2氧化的研究表明, 蛋白质膜红外电化学

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

一种用于小数目造血干细胞的西方印迹协议

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

用斑点印迹分析 A11-positive β淀粉样寡聚物的制备与评价

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial species, the koala (Phascolarctos cinereus), faces prolonged exposure to anthropogenic-induced stressors and assessment of chronic stress in wild populations is urgently warranted. One of the most effective ways to measure chronic stress is through analyzing the glucocorticoid hormone cortisol in hair or fur, as it supports physiological and behavioral responses. This laboratory validation study aims to test current techniques to validate an optimal hormone extraction method to be used as a non-invasive measure of cortisol in koala fur. It is recognized that using non-invasive techniques to measure stress hormones is preferred over traditional, invasive techniques due to their ideal practical and ethical standpoints. Additionally, it is comparatively easier to acquire fur from koalas than it is to acquire samples of their blood. This study used samples of koala fur acquired from the Adelaide Koala and Wildlife Hospital to run a number of hormone extraction techniques in an attempt to validate an optimal cortisol extraction method. Results showed that 100% methanol provided the most optimal solvent extraction compared to 100% ethanol or 100% isopropanol based on parallelism results. In conclusion, this method of cortisol extraction from koala fur provided a reliable non-invasive assay that could be used to study chronic stress in koalas.

Cortisol Measurement in Koala Phascolarctos cinereus Fur

We present a protocol to determine the optimal extraction solvent to measure cortisol from koala fur. The solvents used in this protocol are methanol, ethanol and isopropanol. Determining an optimal extraction solvent will aid in reliably measuring fur to determine the impact of chronic stress on koalas.

科蒂醇测量在考拉 （菲斯科拉托斯西尼鲁斯） 毛皮

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...