Name: co-immunoprecipitation assay for studying functional interactions between receptors and enzymes
Uploaded: 2018-09-28T15:00:00
Description: Watch this Scientific Journal Video about Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes at JoVE.com

该项目由 NIH 赠款1R01AI125640-01 和风湿病研究基金会资助。

1. 细胞转染
<ol><li>种子 HEK 293T 细胞成十二10厘米板 (每板 5 x 106细胞) 转染前的前一天 (图 1)。使用 hemocytometer 执行单元格计数。对于每个盘子, 使用10毫升的 DMEM 补充10% 的血清和1% 青霉素/链霉素。孵化细胞在37°c 在 5% CO2。</li>
	<li>一旦细胞80-90% 汇合, 染 5 HEK 293T 板使用脂基转染试剂与以下质粒之一: SHP2-expressing 向量, PD-1-GFP-expressing 向量 [野生类型的 PD-1 的?...

<ol>
	<li>Montor, W. R., Salas, A., Melo, F. H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+tyrosine+kinases+and+downstream+pathways+as+druggable+targets+for+cancer+treatment:+the+current+arsenal+of+inhibitors.">Receptor tyrosine kinases and downstream pathways as druggable targets for cancer treatment: the current arsenal of inhibitors.</a> Molecular Cancer. 17 (1), (2018).</li><li>Ngoenkam, J., Schamel, W. W., Pongcharoen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+signalling+proteins+recruited+to+the+T-cell+receptor-CD3+complex.">Selected signalling proteins recruited to the T-cell receptor-CD3 complex.</a> Immunology. 153 (1), 42-50 (2018).</li><li>Azoulay-Alfaguter, I., Strazza, M., Pedoeem, A., Mor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coreceptor+programmed+death+1+inhibits+T-cell+adhesion+by+regulating+Rap1.">The coreceptor programmed death 1 inhibits T-cell adhesion by regulating Rap1.</a> Journal of Allergy and Clinical Immunology. 135 (2), 564-567 (2015).</li><li>Chemnitz, J. M., Parry, R. V., Nichols, K. E., June, C. H., Riley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHP-1+and+SHP-2+associate+with+immunoreceptor+tyrosine-based+switch+motif+of+programmed+death+1+upon+primary+human+T+cell+stimulation,+but+only+receptor+ligation+prevents+T+cell+activation.">SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation.</a> The Journal of Immunology. 173 (2), 945-954 (2004).</li><li>Patsoukis, N., 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=Selective+effects+of+PD-1+on+Akt+and+Ras+pathways+regulate+molecular+components+of+the+cell+cycle+and+inhibit+T+cell+proliferation.">Selective effects of PD-1 on Akt and Ras pathways regulate molecular components of the cell cycle and inhibit T cell proliferation.</a> Science Signaling. 5 (230), 46 (2012).</li><li>Pentcheva-Hoang, T., Chen, L., Pardoll, D. M., Allison, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+death-1+concentration+at+the+immunological+synapse+is+determined+by+ligand+affinity+and+availability.">Programmed death-1 concentration at the immunological synapse is determined by ligand affinity and availability.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (45), (2007).</li><li>Yokosuka, T., 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=Programmed+cell+death+1+forms+negative+costimulatory+microclusters+that+directly+inhibit+T+cell+receptor+signaling+by+recruiting+phosphatase+SHP2.">Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2.</a> The Journal of Experimental Medicine. 209 (6), 1201-1217 (2012).</li><li>Hui, E., 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=T+cell+costimulatory+receptor+CD28+is+a+primary+target+for+PD-1-mediated+inhibition.">T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition.</a> Science. 355 (6332), 1428-1433 (2017).</li><li>Sheppard, K. A., 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=PD-1+inhibits+T-cell+receptor+induced+phosphorylation+of+the+ZAP70/CD3zeta+signalosome+and+downstream+signaling+to+PKCtheta.">PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3zeta signalosome and downstream signaling to PKCtheta.</a> FEBS Letters. 574 (1-3), 37-41 (2004).</li><li>Okazaki, T., Maeda, A., Nishimura, H., Kurosaki, T., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1+immunoreceptor+inhibits+B+cell+receptor-mediated+signaling+by+recruiting+src+homology+2-domain-containing+tyrosine+phosphatase+2+to+phosphotyrosine.">PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (24), 13866-13871 (2001).</li><li>Sun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonism+between+binding+site+affinity+and+conformational+dynamics+tunes+alternative+cis-interactions+within+Shp2.">Antagonism between binding site affinity and conformational dynamics tunes alternative cis-interactions within Shp2.</a> Nature Communications. 4, 2037 (2013).</li><li>Peled, M., 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=Affinity+purification+mass+spectrometry+analysis+of+PD-1+uncovers+SAP+as+a+new+checkpoint+inhibitor.">Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a new checkpoint inhibitor.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (3), E468-E477 (2018).</li><li>Jang, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+tyrosine+phosphatase+inhibitor,+pervanadate,+inhibits+angiotensin+II-Induced+beta-arrestin+cleavage.">A protein tyrosine phosphatase inhibitor, pervanadate, inhibits angiotensin II-Induced beta-arrestin cleavage.</a> Molecules and Cells. 28 (1), 25-30 (2009).</li><li>Pluskey, S., Wandless, T. J., Walsh, C. T., Shoelson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+stimulation+of+SH-PTP2+phosphatase+activity+by+simultaneous+occupancy+of+both+SH2+domains.">Potent stimulation of SH-PTP2 phosphatase activity by simultaneous occupancy of both SH2 domains.</a> The Journal of Biological Chemistry. 270 (7), 2897-2900 (1995).</li><li>McAvoy, T., Nairn, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serine/threonine+protein+phosphatase+assays.">Serine/threonine protein phosphatase assays.</a> Current Protocols in Molecular Biology. , (2010).</li><li>Shlapatska, L. M., 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=CD150+association+with+either+the+SH2-containing+inositol+phosphatase+or+the+SH2-containing+protein+tyrosine+phosphatase+is+regulated+by+the+adaptor+protein+SH2D1A.">CD150 association with either the SH2-containing inositol phosphatase or the SH2-containing protein tyrosine phosphatase is regulated by the adaptor protein SH2D1A.</a> The Journal of Immunology. 166 (9), 5480-5487 (2001).</li></ol>

受体-酶相互作用是细胞内信号的关键。许多酶被招募到受体通过 SH2 域绑定到磷酸化酪氨酸, 装饰的尾巴相同的受体。然而, 酶通常被折叠成闭合的非活性构象, 激活需要构象变化11 , 可以由同一受体的其他领域介导。这里描述的化验测量受体和酶之间的相互作用以及这些相互作用引起的活动。
我们使用了一种比色测定法, 利用 p-硝基苯磷酸 (pNPP) 作为基质的 S...

催化受体和受体酪氨酸激酶是跨膜蛋白, 其中细胞外配体的结合导致酶活性的细胞内侧1。有些受体具有受体和酶功能, 而另一些则吸收特定的酶, 如激酶和磷酸的细胞质尾巴。在受体的尾部进行酶的招募, 以及随后这种酶的催化作用, 是两个不同的过程, 不总是由相同的蛋白域2调节。不幸的是, 没有具体的工具来评估相互作用和酶活性的同时。这里描述的功能性免疫沉淀检测是一种很有用的方法, 用于解剖从激活到受体的尾部的酶的招募。这种检测利用免疫沉淀的标记受体的抗体涂层珠。随后对珠进行酶活性测定和印迹分析。这个方法的总目标是发现哪些蛋白质领域是必要的在感受器官和酵素之间的相互作用 (由西部污点分析评估), 并且哪些领域是强制的对酵素的完全活化 (由在珠子测量酶活性测定)。开发用于研究受体相关酶的功能的工具是非常重要的, 因为它们参与了人类疾病的发病机制。此外, 进一步了解这些蛋白质的作用机制可以帮助设计新的治疗干预措施。
程控 death-1 (PD-1) 是 t 细胞表面的抑制受体, 是限制过度 t 细胞反应的必要条件。近年来, anti-PD-1 抗体已被牵连到治疗多发性恶性肿瘤1,2。PD-1 结扎抑制许多 T 细胞功能, 包括增殖, 黏附力和分泌多种细胞因子3,4,5。PD-1 的免疫突触, t 细胞和抗原呈现细胞之间的接口6, 它 colocalizes 与 T 细胞受体 (TCR)7。随后, 酪氨酸磷酸酶 SHP2 [Src 同源 2 (SH2) 域含有酪氨酸磷酸酶 2] 被招募到 PD-1 的细胞质尾部, 导致 TCR 复合体中关键酪氨酸残留物的除磷及其相关的近端信号分子3,4,5,8,9。PD-1 的细胞质尾包含两个酪氨酸图案, 一个 immunoreceptor 酪氨酸的抑制母题 (ITIM) 和一个 immunoreceptor 的酪氨酸基开关的主题 (ITSM)10。两个图案都是磷酸化后 PD-1 结扎9,10。突变研究揭示了 ITSM 在 SHP2 招聘中的主要作用, 而不是 ITIM, 后者在 PD-1 信号和功能方面的作用不清楚4。
SHP2 采用闭合 (折叠), 抑制构象或开放 (延长), 活跃构象11。PD-1 的每个尾部域对 SHP2 绑定或激活的贡献尚未阐明。为了回答这个问题, 我们开发了一种检测方法, 能够对 PD-1 的 SHP2 和它的活动12进行并行测试。我们采用了免疫沉淀和珠粒磷酸酶活性测定法, 同时测试了两者的相互作用和酶活性。使用这一方法, 我们表明, PD-1 的 ITSM 足以招募 SHP2 PD-1 的尾部, 而 ITIM PD-1 是需要充分扩展和激活的酶。
有许多受体有几个相邻的领域在他们的细胞质尾巴。功能性的免疫沉淀分析可以揭示特定领域的作用, 这是必要的蛋白质招募或酶活化。

<table>
	<tbody>
		<tr>
			<td>PBS</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td>Phosphate Buffered Saline</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5424-R</td>
			<td>1.5 ml</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%) Phenol Red</td>
			<td>Gibco</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Denville</td>
			<td>FB5001-H</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Penicillin / Streptomycin</td>
			<td>Fisher</td>
			<td>BP295950</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose without L-glutamine</td>
			<td>Lonza</td>
			<td>BE12-614F</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFect Transfection Reagent</td>
			<td>Qiagen</td>
			<td>301305</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SHP2</td>
			<td>Santa Cruz</td>
			<td>SC-280</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti&#8211;GFP-agarose</td>
			<td>MBL</td>
			<td>D153-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP</td>
			<td>Roche</td>
			<td>118144600</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Actin</td>
			<td>Santa Cruz</td>
			<td>SC-1616</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK-293 cells</td>
			<td>ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>Orthovanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma</td>
			<td>216763</td>
			<td>30%</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td>EDTA-free</td>
		</tr>
		<tr>
			<td>Tris-Glycine SDS Sample Buffer (2X)</td>
			<td>Invitrogen</td>
			<td>LC2676</td>
			<td>Modified Laemmli buffer</td>
		</tr>
		<tr>
			<td>4-20% Tris-Glycine Mini Gels</td>
			<td>Invitrogen</td>
			<td>XP04205BOX</td>
			<td>15-well</td>
		</tr>
		<tr>
			<td>Nitrocellulose membranes</td>
			<td>General Electric</td>
			<td>10600004</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>Sodium chloride</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td>N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Invitrogen</td>
			<td>D1532</td>
			<td>Dithiothreitol</td>
		</tr>
		<tr>
			<td>pNPP</td>
			<td>Sigma</td>
			<td>20-106</td>
			<td>p-Nitrophenyl Phosphate</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045</td>
			<td>Sodium hydroxide</td>
		</tr>
		<tr>
			<td>BCA</td>
			<td>Fisher</td>
			<td>23225</td>
			<td>Bi Cinchoninic Acid assay</td>
		</tr>
	</tbody>
</table>

co-immunoprecipitation assay for studying functional interactions between receptors and enzymes

受体相关酶是细胞活化的主要媒介。这些酵素被调控, 至少部分, 由物理相互作用与受体的细胞质尾巴。这种相互作用经常发生在特定的蛋白质领域, 并导致酶的活化。有几种方法来研究蛋白质之间的相互作用。虽然共同免疫沉淀通常用于研究蛋白质与蛋白质相互作用所需的领域, 但没有化验证明特定领域对所吸收酶的活性的贡献。据此, 本文所描述的方法结合了免疫沉淀和一个珠状酶活性测定, 同时评价蛋白质与相关酶活化的相互作用。本协议的目标是确定对蛋白质和酶之间的物理交互至关重要的领域, 以及对酶的完全活化所必需的域。这一检测的重要性被证明, 因为某些受体蛋白域有助于酶对受体的细胞质尾部的结合, 而其他领域是必要的, 以调节同一酶的功能。

虽然建立了 ITSM PD-1 对 SHP2 约束力的贡献, 但 PD-1 ITIM 的作用不那么明确。由于 SHP2 有两个 SH2 域, 它们可以绑定到 PD-1 上的两个连续 phosphotyrosines (一个在 ITSM 中的一个酪氨酸, 另一个在 ITIM 中), 我们假设 ITSM 的 PD-1 锚 SHP2 到 PD-1, ITIM PD-1 有助于 SHP2 活动, 稳定其打开构象状态11,14。为了测试这一点, 我们开发了一个联合免疫沉淀和?...

Watch this Scientific Journal Video about Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes at JoVE.com

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

免疫沉淀法研究受体与酶的功能相互作用

在这里, 我们提出了一个共同免疫沉淀的协议和一个在珠酶活性检测, 同时研究的具体蛋白质领域的血浆膜受体对酶的招募和酶活性。

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

该方法可以帮助回答细胞信号领域的关键问题，如不同蛋白质域在受体酶相互作用和酶激活中的重要性。该技术的主要优点是，它允许伴随测试受体酶相互作用和这种相互作用对酶活性的功能后果。对于每个板，使用10毫升DMEM，辅以10%FBS和1%青霉素和链霉素。转染前一天，用血细胞计将人类胚胎肾293-T细胞种子放入12个10厘米的细胞中，以计算细胞数。在5°C的37摄氏度下孵育。一旦细胞是80-90%的汇合，使用基于脂质的转染剂转染五个板。按照制造商关于 10 厘米板中粘附细胞的说明执行转染。然后转染相同的第二组五个板，以及一个额外的板，作为非递进的对照，用于在免疫沉淀前测量表达的蛋白质量。在 5%CO2 的组织中培养培养箱中孵育转染的板，48 小时。使用荧光显微镜检查细胞的GFP表达，并确保转染成功发生。将 15 微升 100 毫摩尔正极钠与 50 微升 30% 过氧化氢混合，以准备新鲜的过氧化物混合物。要对PD-1的标记版本进行荧光化，请从PD-1-GFP转染细胞中去除介质。在每个盘子中加入10毫升的普通DMEM和10微升的纯DMEM和10微升的纯DMEM和10微升的纯DMEM。在室温下在黑暗中孵育15分钟。在此之后，每次洗涤使用五毫升 PBS 在冰冷的 PBS 中三次清洗细胞。在进行免疫沉淀时，所有步骤都应在冰上或在四摄氏度下执行。在10毫升缓冲液中溶解一片抑制剂，用蛋白酶抑制剂补充解解缓冲液。还要在用于PD-1-GFP转染板的缓冲液中加入一毫升正极酸钠。向细胞中加入500微升的冰冷解液缓冲液，确保将含有正酸钠的开液缓冲液添加到仅含有PD-1-GFP转染细胞的板中。使用细胞刮刀立即从板中取出和收集细胞。将酸盐转移到1.5毫升的冷管中，并在重力0.005倍和4摄氏度下旋转30分钟。为了从裂酸中收集后核上一提液，在10000倍的重力下旋转它们，在4摄氏度下旋转10分钟。将超自然剂转移到新管中并丢弃颗粒。将第二组六个板的超级钠存放在冰上，供以后进行WCL分析。要开始准备防 GFP 珠子，请先轻轻摇动装有珠子的瓶子，以防止沉淀。每个情况从浆料中去除 40 微升的防 GFP 珠子。在500倍重力和4摄氏度的离心3分钟。取出上流液清洗珠子，确保尽量减少移液器与珠子之间的接触，防止丢失。将珠子重新用 80 微升的解液缓冲液中重新填充。将洗涤的珠子直接从第一组四个板的PD-1-GFP表达细胞中加入细胞。在 0.005 重力下旋转，在 4 摄氏度下旋转 30 分钟，以免疫沉淀 GFP 标记的蛋白质。使用一毫升冷解液缓冲液清洗珠子三次，每次洗涤时不含正交体。然后在2500倍重力下离心10秒。将活性 SHP2 的 lysate 从第一组板中分成三个相等的部分，并在包含带洗涤 PD-1-GFP 的珠子的三个管中各加一部分。将非转染细胞的1/3的酸盐体积添加到野生型PD-1-GFP珠子的第二管中。丢弃剩余的 2/3。在4摄氏度下孵育珠子4小时，在重力的0.005倍下轻轻旋转。在此之后，每次洗涤使用一毫升冷解液缓冲液洗珠子两次。在每个样品中加入80微升的解液缓冲液，确保每个管的总体积为100微升。使用切割移液器尖端，移液器轻轻地向上和向下混合。然后将每个管中的50微升转移到两根新鲜1.5毫升的管子中。用磷酸酶洗涤缓冲液清洗珠子一次。然后完全取出上一液。在珠子中加入100微升的检测缓冲液，在30摄氏度下在温和的搅拌下孵育30分钟。当缓冲液变黄时，通过加入50微升氢氧化钠的摩尔溶液来终止反应。在重力2，500倍时离心10秒。在此之后，将上经器转移到一个96孔板中的两个半面积的井中。读取 405 纳米的吸水剂，将结果表示为 PD-1-GFP 控制野生型版本的相对光学密度。首先在重力2000倍和4摄氏度的温度下旋转珠子30秒，然后取出上一液。加入20微升两次莱姆利缓冲液，在95摄氏度下煮沸5分钟。使用 BCA 试剂盒测量输入控件的蛋白质浓度。将稀释量最大的样品的30微升转移到新管中。使用解液缓冲液将输入控制的其他控制装置稀释到与最稀释的样品相同的浓度。然后将每个稀释的输入控制装置的 30 微升转移到新管中。将相等的体积为莱姆利缓冲液的两倍，煮95摄氏度，5分钟。在此之后，在执行文本协议中概述的西 Blot 分析之前，以 2000 倍的重力和 4 摄氏度旋转珠子 30 秒。在这项研究中，为受体酶相互作用和活化的并行评估，开发了联合免疫沉淀和酶活性测定。毫不奇怪，当 ITSM 发生突变时，SHP2 未能绑定到 PD-1。值得注意的是，ITIM 的突变版本仅抑制了 SHP2 绑定的有限程度。然而，SHP2磷酸酶活性测定表明，ITIM和 ITSM对于酶活性同样不可或缺。这揭示了一个两步激活模型，其中SHP2折叠成一个自动抑制的构象在休息条件下。PD-1 激活后，SHP2 被招募到磷酸化 ITSM 中。但是，ITIM 还必须进行磷酸化，以将 SHP2 展开到其活性构象中。该技术发展后，可为细胞信号领域的研究人员探索蛋白质域在受体-酶相互作用中的功能铺平道路。

Research

JoVE Journal

Immunology and Infection

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

科研

免疫与感染

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

一种新型<em&gt;在体外</em&gt;模型研究人类全血细胞间的相互作用

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

动态粘附法在炎症性肠病抗粘连治疗功能分析中的作用

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

内生菌与人类肠道微生物群使用体外浴发酵系统的相互作用分析

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...