作者感谢埃里克. 芳廷承袭提供了出色的技术援助。这项工作得到了库鲁汶 (格兰特) 的支持。PF/10/018), 全宗与客厅里 Wetenschappelijk Onderzoek (FWO, 授予 no。G. 485.08) 和基金会 Dormeur 瓦杜兹。

1. 细胞培养的维护
注: 1 和2所述的所有步骤均在层流柜的无菌条件下进行。
<ol><li>在37摄氏度和 5% CO2的 T75 培养瓶中生长细胞在一个湿润的孵化器。 注: 在本试验中, 使用 Jurkat 细胞 (即,内在表达 CXCR417) 的人白血病 T 淋巴细胞细胞。CXCR4 在细胞表面的表达应通过流式细胞仪在细胞培养过程中进行评价。但是, 对流式细胞术和试剂的描述, 以确定细胞表面的受体表达水平, 但不属于本协议的范围, 但以前已经描述过17。</li>
	<li>让 Jurkat 细胞在悬浮中生长, 直到它们达到 80-85% 融合。在将细胞传代到新的烧瓶之前, 允许所有试剂达到室温 (RT)。</li>
	<li>添加20毫升新鲜完整的生长培养基 (RPMI-1640 培养基, 10% 胎牛血清 (血清), 2 毫米谷氨酰胺) 到一个新的 T75 培养瓶。</li>
	<li>添加5毫升的 Jurkat 细胞悬浮从原来的 T75 瓶 (包含25毫升细胞悬浮) 到新的 T75 培养瓶。孵育在37°c 和 5% CO2在一个湿润的孵化器。</li>
</ol>2. 制备 CXCL12、化验缓冲器和 Jurkat 细胞进行竞争结合试验。
<ol><li>准备 CXCL12AF647 (20 µg/毫升) 的库存解决方案; 请参阅材料和试剂表) 溶解冻干试剂 (存储在-80 °c, 在黑暗中) 在超纯水补充 0.01% (容量/体积) 吐温20。存储单使用整除数从这个库存解决方案在-80 摄氏度, 保护免受光。</li>
	<li>通过添加40毫升 HEPES (1 米, 20 毫米最终浓度) 到200毫升汉克的平衡盐溶液 (HBSS, 10x, 不含酚红色和不含碳酸氢钠, 1x 最终浓度) 制备化验缓冲器。添加超纯水以获得 2 l 的最终体积, 添加4克 (0.2% 重量/体积) 牛血清白蛋白 (bsa), 并通过磁性搅拌溶解 bsa。最后, 调整 pH 值为 7.4 (使用氢氧化钠) 和过滤溶液通过0.2 µm 毛孔 (参见材料表和试剂) 使用真空歧管。 注: 此检测缓冲器将用于该议定书的所有进一步步骤。</li>
	<li>计算单元格的数量和生存能力。为此, 取一个细胞悬浮的样品和稀释它在磷酸盐缓冲盐水 (PBS)。 注意: 我们经常使用自动细胞生存能力分析仪 (参见材料和试剂表), 能够在不同浓度下计数细胞悬浮液。对于细胞计数, 稀释0.5 毫升细胞悬浮在1.5 毫升 pbs (其他稀释,例如, 0.1 毫升在1.9 毫升 pbs, 也是可能的)。此方法的使用是基于台盼蓝染料排除方法, 以前已经描述了26。还有一些其他的设备在商业上可用于计数细胞数和生存能力, 应该同样良好的工作。</li>
	<li>收集所需的单元格数 (即, ~ 24 x 106在无菌50毫升管中使用一个完整的 96-井板来运行化验结果) 离心 (参见材料和试剂的为离心机的类型) 在 400 x g 为5分钟在rt.</li>
	<li>在不干扰细胞颗粒的情况下, 轻轻地倒上清液。添加新鲜化验缓冲区 (例如, 20 毫升), 并通过轻轻吹打向上和向下并用重悬细胞。</li>
	<li>再次离心细胞在 400 x g 5 分钟的 RT。</li>
	<li>再次倒入上清液, 并在新鲜的化验缓冲器中并用重悬细胞颗粒, 以获得 5 x10 6 细胞/毫升的密度。</li>
</ol>3. 竞争结合试验
注: 实际的竞争结合试验是在 RT 进行的, 可以在无菌条件下进行。
<ol><li>稀释检测缓冲物中所研究的化合物 (见 2.2) 以获得所需浓度。要么准备一个固定浓度的化合物初始筛选 (例如, 10 µM 最终浓度), 或者, 或者, 一个系列稀释系列的浓度, 以更详细的描述化合物 (例如, 1/3, 1/4 或 1/5稀释系列从1µM 开始, 最后集中)。请记住, 复合溶液最终将成为2x 稀释的化验;因此, 准备一个2x 集中解决方案。</li>
	<li>将100µL 的复合溶液 (2x 浓缩) 成一个清晰的96井圆底板 (参见材料目录和试剂表), 按照预定义的实验布局 (例如, 图 1C)。 注: 在这个阶段, 阴性和阳性对照样品包括在化验中。在负控制样本中, 将100µL 的检测缓冲器添加到96井板的井中, 而不是复合物。对于正则控件示例, 在此步骤中还添加了检测缓冲区。另请参见图 1C中的典型实验布局, 其中对几种化合物的稀释系列进行了测试。</li>
	<li>添加50µL 的细胞悬浮 (见 2.7; 0.25 x 106细胞) 从试剂库到96井板使用多通道吸管。在黑暗中在 RT 中孵化出15分钟的盘子。</li>
	<li>添加50µL 的荧光标记为 CXCL12 (即, 100 ng/毫升 CXCL12AF647在化验缓冲区, 4x 浓缩, 25 ng/毫升最终浓度) 从一个类似的试剂库到96井板井。在黑暗中在 RT 孵化30分钟。 注意: 对于负控件示例, 请改用检测缓冲区。因此, 在负控制样本中检测到的荧光信号将对应于 autofluorescent 背景信号 (图 1A)。对于正则控件示例, 请添加50µL CXCL12AF647。阳性控制样本将产生最大荧光信号检测, 因为不包括预先孵化与化合物的潜在抑制 (图 1A)。</li>
	<li>离心96井板在 400 x g 为5分钟在 RT. 通过翻转盘子从颗粒细胞中取出上清。在纸巾上擦干盘子。</li>
	<li>用多通道吸管在试剂库中添加200µL 的新鲜化验缓冲器。立即进行。</li>
	<li>再次离心板5分钟, 在 400 x g 在 RT. 通过翻转盘子, 再将其干燥在组织上, 取出上清液。</li>
	<li>轻轻并用重悬细胞颗粒在200µL 的1% 多聚甲醛溶解在 PBS。此步骤将修复单元格。</li>
	<li>使用流式细胞仪对荧光进行量化, 立即继续该协议。</li>
</ol>4. 流式细胞术对样品的分析
CXCL12AF647染色和固定细胞现在可以使用流式细胞仪进行分析。可以使用几种类型的流 cytometers, 但它们需要配备正确的激光 (即一个红色激光, 励磁范围 ~ 630 nm) 为励磁和适当的过滤器为荧光检测 (发射过滤器 ~ 660 毫微米)。他们需要能够处理样品的96井板格式。在材料和试剂表中给出了合适的流式细胞术装置的例子。
<ol><li>启动设备并打开相应的软件 (请参阅材料和试剂表)。</li>
	<li>选择下面的蜂窝参数以斑点印迹格式进行可视化: 正散射 (FSC)、侧散点 (SSC) 和荧光 (CXCL12AF647) 检测通道。 注: 在 FSC 参数下, 由于检测到的光吸收与细胞直径成正比, 因此根据其大小对细胞进行判别。SSC 参数, 测量在90°角的光散射, 提供了有关细胞粒度的信息。</li>
	<li>选择一个示例 (例如,一个负控制样本), 根据 FSC 和 SSC 参数执行已定义的同质细胞填充的浇口。<ol>
<li>选择自动注射100µL 的固定细胞从这个负控制样本进入流式分析仪。在注入前选择 "混合" 选项, 并使用1.5 µL 的采样流率。</li>
		<li>通过选择 "获取数据" 运行此示例。此示例的 FSC 和 SSC 参数现在将显示在屏幕上。</li>
		<li>选择软件的浇口工具。基于 FSC 和 SSC 斑点杂交可视化, 预先定义了一个均匀和可行的细胞群的门。为此, 请创建一个多边形 (使用软件的浇口工具), 其中包括基于这两个维度的 homogenously 分布式单个单元格 ("事件")。 注: 浇注程序的目的是定义一个均匀和可行的细胞群体, 将用于进一步分析。浇口依赖的假设, 大多数可行的细胞将形成一个同质细胞种群的基础上的 FSC 和 SSC 参数。通过执行这一步骤, 细胞碎片、死细胞和细胞聚集物可以很大程度上被排除在进一步分析之外。在图 1B中给出了浇口过程的说明。</li>
	</ol>
</li>
	<li>选择以分析每个示例的2万个 "事件" (即,单个单元格)。 注意: 这意味着对于每个样本, 在预定义的门内的2万个单元将最终被分析。每个示例的数据获取将继续进行, 直到分析此事件的数量。</li>
	<li>启动运行 (选择 "记录数据")。流式细胞仪现在将通过记录每个样本的平均荧光强度 (多边基金会) 对所有样本进行一次分析。这一多边基金会对应于2万个细胞的平均荧光信号在预定的门之内。</li>
</ol>5. 数据分析
使用为每个样本获得的多边基金会执行所有进一步的计算。对流式细胞仪数据的分析可以通过几个商业上可用的软件包进行 (参见材料和试剂表)。
<ol><li>为了确定荧光结合信号的百分比抑制, 由于复合预孵化, 应用以下公式: <img alt="Equation 1" src="/files/ftp_upload/57271/57271eq1.jpg"> 地方: "多边化" Exp = 实验性 (= 复合处理) 示例的多边化 "多边化" PC = 正控件的多边化 多边化NC = 负控制的多边化</li>
	<li>要确定复合 (即) 的 IC50值, 可将荧光绑定信号减少50% 的化合物的浓度, 请应用以下公式: <img alt="Equation 2" src="/files/ftp_upload/57271/57271eq2.jpg"> 地方: Log轻质. 2 = 一个化合物浓度的对数, 导致不到50% 抑制 PC 和 NC 的多边化值之间的差异 Log轻质 1 = 一个化合物浓度的对数, 导致超过50% 抑制了 PC 和 NC 的多边建筑的价值之间的差异。 注: 另外, 在高活性化合物的情况下, 可以根据每个测试的化合物浓度对应的多边建筑, 生成覆盖数个震级对数的剂量响应曲线。通过使用适当的软件 (参见材料和试剂表) 应用非线性回归曲线, 可以从生成的曲线推断出 IC50值。此分析方法的一个示例显示在图 3中。</li>
</ol>

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G., Nimmagadda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+CXCR4+expression+in+human+cancer+xenografts:+evaluation+of+monocyclam+64Cu-AMD3465.">Imaging CXCR4 expression in human cancer xenografts: evaluation of monocyclam 64Cu-AMD3465.</a> J Nucl Med. 52, 986-993 (2011).</li><li>Hatse, S., Princen, K., Liekens, S., Vermeire, K., De Clercq, E., Schols, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+CXCL12AF647+as+a+novel+probe+for+nonradioactive+CXCL12/CXCR4+cellular+interaction+studies.">Fluorescent CXCL12AF647 as a novel probe for nonradioactive CXCL12/CXCR4 cellular interaction studies.</a> Cytometry A. 61, 178-188 (2004).</li><li>Wootten, D., Christopoulos, A., Sexton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+paradigms+in+GPCR+allostery:+implications+for+drug+discovery.">Emerging paradigms in GPCR allostery: implications for drug discovery.</a> Nat Rev Drug Discov. 12, 630-644 (2013).</li><li>Louis, K. S., Siegel, 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=Cell+viability+analysis+using+trypan+blue:+manual+and+automated+methods.">Cell viability analysis using trypan blue: manual and automated methods.</a> Methods Mol Biol. 740, 7-12 (2011).</li><li>Perry, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maraviroc:+a+review+of+its+use+in+the+management+of+CCR5-tropic+HIV-1+infection.">Maraviroc: a review of its use in the management of CCR5-tropic HIV-1 infection.</a> Drugs. 70, 1189-1213 (2010).</li><li>Moyle, G., DeJesus, E., Boffito, M., Wong, R. S., Gibney, C., Badel, K., MacFarland, R., Calandra, G., Bridger, G., Becker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proof+of+activity+with+AMD11070,+an+orally+bioavailable+inhibitor+of+CXCR4-tropic+HIV+type+1.">Proof of activity with AMD11070, an orally bioavailable inhibitor of CXCR4-tropic HIV type 1.</a> Clin Infect Dis. 48, 798-805 (2009).</li><li>Balabanian, K., Lagane, B., Infantino, S., Chow, K. Y., Harriague, J., Moepps, B., Arenzana-Seisdedos, F., Thelen, M., Bachelerie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+SDF-1/CXCL12+binds+to+and+signals+through+the+orphan+receptor+RDC1+in+T+lymphocytes.">The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes.</a> J Biol Chem. 280, 35760-35766 (2005).</li><li>Burns, J. M., Summers, B. C., Wang, Y., Melikian, A., Berahovich, R., Miao, Z., Penfold, M. E., Sunshine, M. J., Littman, D. R., Kuo, C. J., Wei, K., McMaster, B. E., Wright, K., Howard, M. C., Schall, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chemokine+receptor+for+SDF-1+and+I-TAC+involved+in+cell+survival,+cell+adhesion,+and+tumor+development.">A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development.</a> J Exp Med. 203, 2201-2213 (2006).</li><li>Stoddart, L. A., Kilpatrick, L. E., Briddon, S. J., Hill, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+pharmacology+of+G+protein-coupled+receptors+with+fluorescent+ligands.">Probing the pharmacology of G protein-coupled receptors with fluorescent ligands.</a> Neuropharmacology. 98, 48-57 (2015).</li><li>Vernall, A. J., Hill, S. J., Kellam, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+small-molecule+fluorescent-conjugate+toolbox+for+Class+A+GPCRs.">The evolving small-molecule fluorescent-conjugate toolbox for Class A GPCRs.</a> Br J Pharmacol. 171, 1073-1084 (2014).</li><li>Stoddart, L. A., White, C. W., Nguyen, K., Hill, S. J., Pfleger, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-+and+bioluminescence-based+approaches+to+study+GPCR+ligand+binding.">Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding.</a> Br J Pharmacol. 173, 3028-3037 (2016).</li></ol>

作者没有什么可透露的。

与其他类型的结合分析 (即,饱和结合和动力学结合实验) 相比, 竞争结合检测最适合于筛选目的。事实上, 它们允许对大量未标记化合物 (例如小分子) 进行评估, 通过评分它们的能力来干扰固定数量的标记受体配体的绑定。与标记配体绑定到其他受体部位的化合物在化验中可能仍未被发现。虽然本文所描述的竞争约束实验尤其侧重于趋化因子受体 CXCR4, 但它也可能成为其他受体竞争结合分析?...

G 蛋白耦合受体 (受体) 是细胞表面蛋白, 可由胞外配体 (例如,肽, 蛋白质激素, 胺) 激活, 从而调节许多生理和发育过程1。当激动剂占据其 GPCR 结合袋, 诱导构象变化的受体蛋白促进与细胞内受体相关的 heterotrimeric g 蛋白的结合, 由 gα-GDP 和 gβγ亚基组成。随后, gα亚基的 GDP 交换 GTP 导致 g 蛋白亚基 (gα-GTP 和 gβγ) 的离解, 进而将进一步启动下游信号通路23。当 gα-GTP 水解后, gα-GDP 和 Gβγ亚基的重新组合将 g 蛋白转换回休眠状态3,4。不同类型的 g 蛋白存在 (gs, g i/o,gq, g12/13), 这些分类基于序列相似性与 Gα亚基5。所有这些 G 蛋白诱导定义的细胞内信号通路的基础上的生物反应的受体活化。受体活化后, GPCR 激酶 (GRKs) phosphorylate 受体的细胞内尾部, 从而促进与β arrestins 的相互作用。这一过程导致 G 蛋白信号的终止, 受体脱敏和内部化6。β-arrestins 也是多分子复合体的一部分, 触发信号叶栅独立于 G 蛋白信号7。
受体是治疗干预的最有效的分子靶点之一, 因为解除 GPCR 介导的信号, 例如由于受体基因或受体过度表达的功能增益突变, 导致许多人类疾病8。因此, 受体代表了制药行业调查的最重要的药物靶类之一8,9,10。临床相关 GPCR 的一个显著例子是 CXC 趋化因子受体 4 (CXCR4), 它可以由唯一的天然配体激活, CXC 趋化因子配体 12 (CXCL12)11。由于其作为人体免疫机能丧失病毒 1 (HIV-1) 进入和感染的主要联合受体在分化 4 (CD4) 阳性 T 淋巴细胞12中已确立的作用, CXCR4 首先被调查为抗病毒药物靶。CXCL12-CXCR4 在骨髓中的相互作用进一步调节茎和祖细胞的保留和归巢13。此外, 考虑到它参与癌症生物学的许多方面 (例如,肿瘤细胞存活, 转移, 肿瘤相关血管生成)14和其他一些人类疾病 (例如,炎症疾病)15, CXCR4作为药物发现的一个有希望的目标, 引起了极大的兴趣。AMD3100, 一个专门针对 CXCR4 的小分子, 最初被发现为抗 HIV 药物候选16 , 仍然是迄今为止17所描述的最有效的 CXCR4 拮抗剂之一。然而, 它作为抗病毒药物的发展已停止了18。目前该分子作为干细胞动员剂在治疗多发性骨髓瘤和淋巴瘤患者中的应用18。其他一些化学无关的小分子和生物制剂, 抑制 CXCR4 功能的不同效力被描述为19。
受体结合方法是药理学中有价值的工具, 可以识别与 GPCR 直接作用的化合物 (例如,小分子)。为了进行具有约束力的研究, 不需要事先了解一个给定 GPCR 的胞内信号特性或功能。虽然这可以被认为是一个优势, 它意味着可以证明受体结合的化合物需要进一步的特点, 评估其潜在的竞赛或拮抗活性。这项活动可以用药理学或生物化验方法来评估, GPCR 正在研究中。依赖于它们的活动剖面, 受体结合分子可能会进化成为新的铅化合物, 用于研究前临床和临床研究。分子, 专门绑定到一个高亲和力的受体也可以作为支架, 以产生治疗或诊断工具, 例如通过 radiolabeling 他们为无创性的在体内成像肿瘤细胞20, 或作为潜在的有针对性地提供治疗方法的车辆21。在 CXCR4 的情况下, 肿瘤细胞的体内成像已经用老鼠模型证明了, 其中标记了 CXCR4-targeting 分子允许人类癌症的可视化20,22,23.
在本报告中, 我们描述了一项竞争结合试验的详细协议, 它能够识别直接干扰激动剂 (CXCL12) 与 CXCR4 结合的小分子和生物制剂。该检测的基本原理是在固定量的荧光标记配体之间的竞争 (CXCL12AF647, 参见材料和试剂表) 和未标记的化合物绑定到受体蛋白17,24. 然后用流式细胞仪分析标记配体与单细胞表达 CXCR4 的特异荧光信号。当未标记的小分子破坏 CXCL12AF647和 CXCR4 之间的交互时, 此荧光信号将会减少。该化验使用非操作的活细胞内在表达 CXCR4 (即, Jurkat 细胞)。因此, 不需要细胞膜的准备, 这使得化验方便, 快速, 并与增加的吞吐量兼容。由于使用了荧光标记配体, 放射性被避免。
由于 CXCL12 是 CXCR4 的自然激动剂, 因此干扰 CXCL12AF647绑定的小分子化合物很可能与 orthosteric 受体结合站点 (即,由自然激动剂)。如果它们不影响 CXCL12 的束缚, 那么与 orthosteric 结合部位地形不同的受体结合部位的分子将保持不被察觉。例如, 积极和消极的构调制器, 一个重要的和新兴的类别的 GPCR 靶向分子作用于构结合点25, 将有可能不会被拿起这个化验。此外, 是否有这种结合的检测功能的化合物, 作为受体拮抗剂或兴奋剂不能获得。因此, 需要对所鉴定的化合物进行其他药理学或功能受体相关化验。这些化验可能包括 (组合) 细胞荧光或基于发光的检测第二信使 (例如, Ca2 +, 循环腺苷磷酸 (阵营)), 表型或生物测定和β arrestin招聘分析的选择取决于研究中 GPCR 的具体信号特性。因此, 本文所描述的竞争性结合试验主要是一种初步筛选化验, 需要辅以其他细胞化验, 以使化合物具有受体结合效力的深入描述。

<table border="0" cellpadding="0" cellspacing="0" width="1217">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">BD FACSCanto II</td>
			<td style="width:247px;">Becton Dickinson</td>
			<td style="width:281px;">Not applicable</td>
			<td style="width:304px;">Flow cytometry device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">BD FACSDIVA Software</td>
			<td style="width:247px;"></td>
			<td style="width:281px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">BD FACSArray</td>
			<td style="width:247px;">Becton Dickinson</td>
			<td style="width:281px;">Not applicable</td>
			<td>Flow cytometry device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">BD FACSArray System Software</td>
			<td style="width:247px;"></td>
			<td style="width:281px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Graphpad Prism</td>
			<td style="width:247px;">Graphpad</td>
			<td style="width:281px;"></td>
			<td>software package used for nonlinear regression analysis in Figure 2 and Figure 3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:387px;">FlowJo</td>
			<td style="width:247px;">FlowJo is now a wholly owned subsidiary of BD.</td>
			<td style="width:281px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Vi-CELL</td>
			<td style="width:247px;">Beckman Coulter</td>
			<td style="width:281px;">Not applicable</td>
			<td>cell viability analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Sigma 3-18 KS</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:281px;">Not applicable</td>
			<td>centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">AMD3100</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:281px;">A5602-5mg</td>
			<td>specific CXCR4 antagonist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Maraviroc</td>
			<td style="width:247px;">Pfizer</td>
			<td style="width:281px;"></td>
			<td>antiretroviral drug, CCR5 antagonist, available for research at Selleckchem (cat#S2003), Sigma (cat#PZ0002)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">h-SDF1a (AF647)</td>
			<td style="width:247px;">ALMAC</td>
			<td style="width:281px;">CAF-11-B-01</td>
			<td>fluorescently labeled CXCL12, CXCL12AF647</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:247px;">Gibco (Life Technologies)</td>
			<td style="width:281px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:281px;">A1933-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">HBSS (10x), calcium, magnesium, no phenol red</td>
			<td style="width:247px;">Gibco (Life Technologies)</td>
			<td style="width:281px;">14065-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">HEPES (1M)</td>
			<td style="width:247px;">Gibco (Life Technologies)</td>
			<td style="width:281px;">15630-056</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td style="width:247px;">Gibco (Life Technologies)</td>
			<td style="width:281px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Jurkat cells</td>
			<td style="width:247px;">ATCC</td>
			<td style="width:281px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Reagent reservoir PP</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:281px;">BR703411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rapid flow filter: 0.2 &#181;m aPES</td>
			<td style="width:247px;">Thermo Scientific</td>
			<td>566-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Sterilin microtiter plate, 96-well, U bottom, clear</td>
			<td style="width:247px;">Thermo Scientific</td>
			<td style="width:281px;">611U96</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Falcon tubes, 50ml</td>
			<td style="width:247px;">Greiner Bio-One</td>
			<td style="width:281px;">227 261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Tissue culture flask (T75)</td>
			<td style="width:247px;">Corning</td>
			<td style="width:281px;">353024</td>
			<td></td>
		</tr>
	</tbody>
</table>

a flow cytometry-based assay to identify compounds that disrupt binding of fluorescently-labeled cxc chemokine ligand 12 to cxc chemokine receptor 4

药物靶向 G 蛋白偶联受体 (受体) 是非常重要的人类健康, 因为功能失调的 GPCR 介导信号有助于许多疾病的进展。配体/受体对 CXC 趋化因子配体 12 (CXCL12)/CXC 趋化因子受体 4 (CXCR4) 引起了重要的临床兴趣, 例如作为治疗癌症和炎症性疾病的潜在目标。因此, 小分子以及专门针对 CXCR4 和抑制受体功能的治疗性抗体被认为是宝贵的药理工具。本文介绍了一种基于流式细胞仪的细胞检测方法, 它可以识别出将 CXCL12 绑定到 CXCR4 的化合物 (例如,小分子)。从根本上说, 该检测依赖于固定量的荧光标记为 CXCL12, CXCR4 的自然趋化因子激动剂和未标记化合物之间的受体结合的竞争。因此, 本试验避免了放射性标记探针的不良使用。此外, 活细胞被用作受体 (CXCR4) 的来源, 而不是细胞膜的准备。这样就可以方便地将检测方法与平板格式进行调整, 从而提高了吞吐量。这种检测已被证明是一种有价值的通用药物发现检测, 以确定 CXCR4-targeting 化合物。该协议可能会适应其他受体, 至少如果荧光标记配体可用或可以生成。对于在激活这些受体时诱导的细胞内信号通路的先验知识是不需要的。

绑定分析的一般工作流显示在图 1A中。在图 1B中描述了在标准实验中为不同样本类型获得的流式细胞仪数据的类型 (即负控制、正控制和实验样本), 以及可能的版面布局来执行96井板格式的化验在图 1C中给出。Jurkat 细胞的孵化, 内在表达趋化因子受体 CXCR4 (即,这一检测中的兴趣 GPCR), 随着荧光?...

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A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

本文介绍了一种基于流式细胞术的细胞结合检测方法, 主要用作筛选工具, 用于识别抑制荧光标记为 CXC 趋化因子配体 12 (CXCL12) 的化合物对 CXC 趋化因子受体 4 (CXCR4) 的束缚。

一种基于流式细胞仪的检测方法, 用于识别破坏荧光标记的 CXC 趋化因子配体12到 CXC 趋性因子受体4的化合物

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

a-flow-cytometry-based-assay-to-identify-compounds-that-disrupt

Research

JoVE Journal

Biology

13.6K 次观看.  KU Leuven. 本文介绍了一种基于流式细胞术的细胞结合检测方法, 主要用作筛选工具, 用于识别抑制荧光标记为 CXC 趋化因子配体 12 (CXCL12) 的化合物对 CXC 趋化因子受体 4 (CXCR4) 的束缚。

视频: A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

科研

生物学

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

类固醇受体的生化重建•Hsp90的蛋白质复合物和配体激活绑定

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

流式细胞仪分离纯化<em&gt; S.酵母</em&gt;合子

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

生产<em&gt;热带爪蟾</em&gt;卵提取物识别微管相关的RNA

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

非洲爪蟾作为一种模型来确定翻译减值

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯:天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...