Name: sorla and clc:clf-1-dependent downregulation of cntfrα as demonstrated by western blotting, inhibition of lysosomal enzymes, and immunocytochemistry
Uploaded: 2017-01-06T10:45:00.000+00:00
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Description: Watch this Scientific Journal Video about SorLA and CLC:CLF-1-dependent Downregulation of CNTFRÎ± as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry at JoV

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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。

<table><tbody><tr><td>Zeocin</td><td>InvivoGen</td><td>ant-zn-1</td><td></td></tr><tr><td>Hygromycin B Gold</td><td>InvivoGen</td><td>ant-hg-1</td><td></td></tr><tr><td>FuGENE 6 Transfection Reagent&amp;nbsp;</td><td>Roche</td><td>11 815 091 001</td><td></td></tr><tr><td>4-well plates (Nunclon Delta Surface)</td><td>Thermo Scientific</td><td>176740</td><td></td></tr><tr><td>Safe-Lock tubes</td><td>Eppendorf</td><td>0030 120.086</td><td></td></tr><tr><td>Dulbecco Modified Eagle&amp;acute;s Medium (DMEM)</td><td>Lonza</td><td>BE12-604F/U1</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Thermo Scientific</td><td>10270106</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Scientific</td><td>15140122</td><td></td></tr><tr><td>Dulbecco Phosphate Buffered Saline&amp;nbsp; (DPBS)</td><td>HyClone</td><td>SH30028.02</td><td></td></tr><tr><td>CLC:CLF-1</td><td>R&amp;amp;D Systems</td><td>1151-CL/CF</td><td></td></tr><tr><td>Mouse anti-LAMP-1&amp;nbsp;</td><td>DSHP</td><td>H4A3</td><td></td></tr><tr><td>Rabbit anti-sorLA</td><td></td><td></td><td>Reference 16</td></tr><tr><td>Mouse anti-b-actin&amp;nbsp;</td><td>Sigma</td><td>A2228</td><td></td></tr><tr><td>Mouse anti-Myc</td><td>Genscript</td><td>A00704</td><td></td></tr><tr><td>Goat anti-CNTFRa&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>AF-303</td><td></td></tr><tr><td>Rabbit anti-pSTAT3&amp;nbsp;</td><td>Cell signaling Technology</td><td>9145s</td><td></td></tr><tr><td>Mouse anti-STAT3&amp;nbsp;</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></tr><tr><td>Anti-goat HRP-linked antibody</td><td>Dako</td><td>P0160</td><td></td></tr><tr><td>Donkey anti-goat secondary antibody, Alexa Fluor 488 conjugate</td><td>Invitrogen</td><td>A11055</td><td></td></tr><tr><td>Donkey anti-mouse secondary antibody, Alexa Fluor 568 conjugate</td><td>Invitrogen</td><td>A10037</td><td></td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>L8511-5MG</td><td></td></tr><tr><td>Pepstatin A&amp;nbsp;</td><td>Sigma</td><td>P4265-5MG</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>AppliChem</td><td>A1388</td><td></td></tr><tr><td>Tris-HCl</td><td>Merck</td><td>1,082,191,000</td><td></td></tr><tr><td>EDTA:Titriplex III</td><td>Merck</td><td>1,084,180,250</td><td></td></tr><tr><td>cOmplete mini EDTA-free</td><td>Roche</td><td>11836170001</td><td></td></tr><tr><td>PhosSTOP</td><td>Roche</td><td>04 906 837 001</td><td></td></tr><tr><td>LDS sample Buffer (4x)</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 (PFA)</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></tbody></table>

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 CNTFRÎ± 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

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Biochemistry

7.3K 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

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

科研

癌症研究

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

遗传学

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

一种检测慢性淋巴细胞白血病新 nfat2 靶基因的染色质免疫沉淀法

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

原发性慢性淋巴细胞白血病细胞的细胞次细胞分段监测核/细胞质蛋白贩运

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

神经科学

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

适配器，网格蛋白相互作用在体内和体外研究

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

生物学

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

活细胞钙成像相结合的siRNA介导的基因沉默标识CA 2 +泄漏通道内质网膜和监管机制

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

<meta charset="utf8"></head><body>基于 FRET 的无电生理学监测 LRRC8 通道活性的方法

Monitoring Leucine-Rich Repeat Containing 8 Channel (LRRC8/VRAC) Activity Using Sensitized-Emission F&#246;rster Resonance Energy Transfer (SE-FRET)

Members of the LRRC8 protein family form heteromeric ion and osmolyte channels with roles in numerous physiological processes. As volume-regulated anion channels (VRACs)/volume-sensitive outwardly rectifying channels (VSORs), they are activated upon osmotic cell swelling and mediate the extrusion of chloride and organic osmolytes, leading to the efflux of water and hence cell shrinkage. Beyond their role in osmotic volume regulation, VRACs have been implicated in cellular processes such as differentiation, migration, and apoptosis. Through their effect on membrane potential and their transport of various signaling molecules, leucine-rich repeat containing 8 (LRRC8) channels play roles in neuron-glia communication, insulin secretion, and immune response. The activation mechanism has remained elusive. LRRC8 channels, like other ion channels, are typically studied using electrophysiological methods. Here, we describe a method to detect LRRC8 channel activation by measuring intra-complex sensitized-emission F&#246;rster resonance energy transfer (SE-FRET) between fluorescent proteins fused to the C-terminal leucine-rich repeat domains of LRRC8 subunits. This method offers the possibility to study channel activation in situ without exchange of the cytosolic environment and during processes such as cell differentiation and apoptosis.

<meta charset="utf8"></head><body>使用敏化发射 Förster 共振能量转移 （SE-FRET） 监测含有 8 通道亮氨酸的富含亮氨酸重复序列 （LRRC8/VRAC） 活性

Electrophysiology is the gold standard for investigating ion channel activity. However, there are plenty of alternative approaches, including optical methods. Here, we describe a method to monitor the activity of the leucine-rich repeat containing 8 channel (LRRC8)-formed anion channels using an inter-subunit F&#246;rster resonance energy transfer (FRET)-based method.

使用敏化发射 Förster 共振能量转移 （SE-FRET） 监测含有 8 通道亮氨酸的富含亮氨酸重复序列 （LRRC8/VRAC） 活性

Members of the LRRC8 protein family form heteromeric ion and osmolyte channels with roles in numerous physiological processes. As volume-regulated anion ...

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

摘要

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此视频中的章节

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

CRISPR 的染色质环结构重组

一种检测慢性淋巴细胞白血病新 nfat2 靶基因的染色质免疫沉淀法

原发性慢性淋巴细胞白血病细胞的细胞次细胞分段监测核/细胞质蛋白贩运

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

适配器，网格蛋白相互作用在体内和体外研究

活细胞钙成像相结合的siRNA介导的基因沉默标识CA^{2 +}泄漏通道内质网膜和监管机制

使用敏化发射 Förster 共振能量转移（SE-FRET）监测含有 8 通道亮氨酸的富含亮氨酸重复序列（LRRC8/VRAC）活性

使用敏化发射 Förster 共振能量转移（SE-FRET）监测含有 8 通道亮氨酸的富含亮氨酸重复序列（LRRC8/VRAC）活性

使用敏化发射 Förster 共振能量转移（SE-FRET）监测含有 8 通道亮氨酸的富含亮氨酸重复序列（LRRC8/VRAC）活性

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

摘要

探索更多视频

此视频中的章节

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

CRISPR 的染色质环结构重组

一种检测慢性淋巴细胞白血病新 nfat2 靶基因的染色质免疫沉淀法

原发性慢性淋巴细胞白血病细胞的细胞次细胞分段监测核/细胞质蛋白贩运

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

适配器，网格蛋白相互作用在体内和体外研究

活细胞钙成像相结合的siRNA介导的基因沉默标识CA 2 +泄漏通道内质网膜和监管机制

使用敏化发射 Förster 共振能量转移 （SE-FRET） 监测含有 8 通道亮氨酸的富含亮氨酸重复序列 （LRRC8/VRAC） 活性

使用敏化发射 Förster 共振能量转移 （SE-FRET） 监测含有 8 通道亮氨酸的富含亮氨酸重复序列 （LRRC8/VRAC） 活性

使用敏化发射 Förster 共振能量转移 （SE-FRET） 监测含有 8 通道亮氨酸的富含亮氨酸重复序列 （LRRC8/VRAC） 活性

活细胞钙成像相结合的siRNA介导的基因沉默标识CA^{2 +}泄漏通道内质网膜和监管机制

使用敏化发射 Förster 共振能量转移（SE-FRET）监测含有 8 通道亮氨酸的富含亮氨酸重复序列（LRRC8/VRAC）活性

使用敏化发射 Förster 共振能量转移（SE-FRET）监测含有 8 通道亮氨酸的富含亮氨酸重复序列（LRRC8/VRAC）活性

使用敏化发射 Förster 共振能量转移（SE-FRET）监测含有 8 通道亮氨酸的富含亮氨酸重复序列（LRRC8/VRAC）活性