作者承认来自 NIH/研究院 (R01GM084135 至 HJN) 和弘水谷基金会 Glycoscience (授予 #110071 HJN) 的支持, 并感谢汤姆五. 李的讨论和建议的化验, 斯比洛斯 Artavanis, Tsakonas, 雨果 Bellen,罗伯特弗莱明, 肯欧文并且果蝇基因组学资源中心 (DGRC) 为质粒和细胞线。

1. 为欺诈击倒的双绞 RNA (dsRNA) 的制备
<ol>
	<li>产品的 PCR 扩增
	<ol>
		<li>使用野生类型的黄白色(y w) 基因组 DNA 和pAc5.1-EGFP作为模板和下面的入门对来放大 dsRNA 合成中使用的 DNA 片段。使用以下 PCR 热剖面: 变性 (95 ° c, 三十年代), 退火 (58 ° c, 三十年代) 和延长 (72 ° c, 1 min)。 
		增强型绿色荧光蛋白(EGFP) dsRNA 底漆 (5 "-3")- 正向引物-GAAATTAATACGACTCACTATAGGGGGTGAGCAAGGGCGAGGAG 反向底漆-GAAATTAATACGACTCACTATAGGGGGTCTTTGCTCAGGGCGG 
		dsRNA 底漆 (5 "-3")- 正向引物-TTAATACGACTCACTATAGGGGAGATGCTGTATGTGGACACGGAT 反向底漆-TTAATACGACTCACTATAGGGGAGATCCGTGGATAACCTTAACGA 注意: EGFP dsRNA 用作负控制。</li>
	</ol>
	</li>
	<li>根据制造商的协议, 用商业凝胶纯化试剂盒凝胶纯化聚合酶链反应 (PCR) 产品。</li>
	<li>根据制造商的协议, 使用能够从 T7 启动子转录的商用产品进行体外转录。</li>
	<li>根据制造商的协议和存储在-80 ° c, 纯化 dsRNA 使用 RNA 提纯试剂盒。</li>
	<li>使用以下步骤评估欺诈击击的效率 (1.5. 1-1. 5.5). 
	<ol>
		<li>使用例和板 5 x 105 S2 单元, 在1毫升/施耐德培养基中的6个井板的每一个井中, 加上10% 胎牛血清 (FBS) 和100的 U/毫升青霉素-链霉素, 对这些细胞进行手工计数。</li>
		<li>添加7.5 µg 的EGFP-或欺诈 dsRNA 每个井和孵化在25° c 24 小时. </li>
		<li>获取控制 (EGFP dsRNA 处理) 和欺诈 dsRNA 处理的 S2 细胞由温和的移, 然后在 1000 x g 的离心化, 在25° c 5 分钟, 并通过 rna 提取试剂盒的 rna 分离过程, 根据制造商的协议。</li>
		<li>利用 qPCR 试剂和底漆/探针和以下 pcr 热剖面 (95 ° c, 十五年代) 和退火/延长 (60 ° c, 1 分钟), 用分光光度计和过程100的 rna 对 rna 进行量化, 用于1步 qRT pcr。 注: 请参见材料表, 以获取有关入门/探头集和用于欺诈的仪器的信息, 并在这些研究中控制 qRT PCR 实验. </li>
		<li>使用 2 -ΔΔCT方法16计算相对欺诈 mRNA 级别。</li>
	</ol>
	</li>
</ol>
2. 评价切口受体与反式配体之间的结合
<ol>
	<li>准备信号接收细胞 (S2 细胞表达缺口受体)。
	<ol>
		<li>使用例和板 5 x 105 S2 和稳定的 S2-Notch 单元, 在1毫升/好的施耐德培养基中加上 10% FBS 和 100 U/毫升青霉素-链霉素, 以手动计数单元格。 注: 对于 S2-Notch 细胞, 可从果蝇基因组资源中心 (DGRC), 添加 200 nM 甲氨蝶呤在媒体。准备一个0.5 毫米的甲氨蝶呤库存解决方案, 首先准备20毫米甲氨蝶呤溶液250µL 1 米氢氧化钠。其次, 稀释此溶液为0.5 毫米在1米磷酸盐缓冲盐水和商店在-20 ° c。在 S2-Notch 细胞中, 缺口蛋白的表达在pMT向量中由果蝇金属硫蛋白启动子控制。</li>
		<li>每井加7.5 µg dsRNA, 在25° c 下孵育24小时。</li>
		<li>添加 0.7 mM 丘索4以诱导凹槽的表达, 并在25° c 下孵育3天。 注: 丘索4用于诱导金属硫蛋白启动子控制的蛋白质表达。</li>
	</ol>
	</li>
	<li>准备信号发送细胞 (S2 细胞表达三角洲或有锯齿配体)。
	<ol>
		<li>使用例和板〜 5 x 106稳定 S2-Delta 或 S2-SerrateTom单元格, 在1毫升/好的施耐德培养基中补充 10% FBS 和 100 U/毫升青霉素-链霉素, 以手动计数单元格。 注: 添加 200 nM 甲氨蝶呤为 S2-Delta 细胞和100µg/毫升霉 S2-Serrate汤姆细胞在媒体。三角洲和锯齿的表达式汤姆-一个番茄标记, 功能版本有锯齿的12-在pMT向量中的果蝇金属硫蛋白启动子下。</li>
		<li>添加 0.7 mM 丘索4以诱导配体的表达, 并在25° c 下孵育3小时。</li>
	</ol>
	</li>
	<li>执行信号发送和信号接收单元之间的聚合。
	<ol>
		<li>用温和的移和平板 2.5 x 105细胞 dsRNA 处理的 S2 (控制) 和 S2-Notch 细胞, 在24井板上通过手工计数例。</li>
		<li>将 5 x 105稳定 S2-Delta 或 S2-SerrateTom单元格添加到施耐德培养基的200µL 的总容量中 (辅以 10% FBS 和100的 U/毫升青霉素-链霉素)。 注: 所有 S2 细胞处理 (包括电镀、诱导和 dsRNA 处理) 在生物安全柜下进行。</li>
		<li>将盘放在 150 rpm (942.48 rad/分钟) 的轨道振动筛上。</li>
		<li>1分钟后, 将每一个井的内容混合, 取20µL, 计算骨料数。<ol>
			<li>同时采用10x 放大 (PLL 10/0.25 目标) 在倒置复合显微镜下进行具有代表性的图像。</li>
			<li>使用例手动计数聚合 (和 #62; 6 单元格)。</li>
		</ol>
		</li>
		<li>把盘子放在振动筛上。</li>
		<li>重复图像采集和计数后5分钟和15分钟的聚合。</li>
	</ol>
	</li>
	<li>执行反绑定的量化。
	<ol>
		<li>计算 S2 单元格和 S2-Delta 或 S2-SerrateTom单元格之间的每个 mL 的聚合数, 作为后台控件。</li>
		<li>计算 S2-Notch 和 S2-Delta 或 S2-SerrateTom单元格之间每毫升的聚合数 (跨绑定的大小)。</li>
	</ol>
	</li>
</ol>
3。由顺式配体对缺口与反式配体结合的抑制作用的评价
<ol>
	<li>准备信号接收单元。
	<ol>
		<li>板 5 x 105 S2 细胞在6井板的每井1毫升/好的施耐德培养基补充 10% FBS 和 100 U/毫升青霉素-链霉素。</li>
		<li>每井加7.5 µg dsRNA, 在25° c 下孵育24小时。</li>
		<li>用pBluescript和pmt-缺口11 (DGRC) 单独或与pmt-凹槽和pmt-增量11 (DGRC) 或pmt-锯齿 17共用 dsRNA 处理过的单元格根据制造商的协议, 使用商业转染试剂, 2 µg/井的 DNA 浓度。 注意: pBluescript用作控件。co-transfected 细胞将被称为 S2-Notch 和 #38;D elta瞬态或 S2-Notch 和 #38; 有锯齿的瞬态单元格。</li>
		<li>在25° c 下孵育瞬时转染的 S2 细胞24小时。</li>
		<li>添加 0.7 mM 丘索4以诱导切口和配体的表达, 并在25° c 下孵育3天。</li>
	</ol>
	</li>
	<li>准备信号发送单元, 如2.2 所述。</li>
	<li>如2.3 节所述, 执行信号发送和信号接收单元之间的聚合。</li>
	<li>通过cis配体对反式绑定进行量化抑制。
	<ol>
		<li>在 S2 单元格和 S2-Delta 或 S2-Serrate 单元格之间计算每个 mL 的聚合数作为背景控件。</li>
		<li>通过以下方式量化cis-配体的抑制程度。<ol>
			<li>将 A定义为 S2-Notch瞬态和 S2-Delta 单元格之间的聚合数。</li>
			<li>将B定义为 S2 单元格之间的聚合数, 瞬时 co-transfected 与凹槽和 Delta (S2-Notch 和 #38;D elta瞬态) 和 S2-Delta 单元格之间。</li>
			<li>计算相对聚合C = (B x 100)/A</li>
			<li>用cis-配体 (δ或有锯齿) 作为 100- C计算抑制的大小。 注意: 举个例子, 如果相对聚合为 45%, 则cis-配体被认为是抑制跨绑定的55%。</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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作者没有利益冲突。

标准缺口信号依赖于缺口受体与它的配体5之间的相互作用。虽然大多数研究的缺口通路主要考虑到缺口和配体的结合在相邻的细胞 (跨), 缺口和相同细胞配基确实相互作用, 这些 so-called 的cis-相互作用可以发挥抑制作用的缺口信号3,4。因此, 要破译修饰符对标准缺口信号的影响的机制, 通常要检查两种类型的缺口配体相互作用 (跨和cis)。然而, 在许多情况下, 在动物的发展, 参与这两种相互作用和他们的反馈调节复杂的在体内研究1,6。在本议定书中, 我们提供了一个详细的方法,体外细胞聚集法使用果蝇S2 细胞来评估缺口受体与反式配基的结合及其抑制cis配.我们已经介绍了这种方法的利用, 以评估一个基因修饰的缺口信号 (xylosyltransferase 欺诈的7,8) 对受体-配体结合的影响。
首先, 在稳定 的 S2-Delta 和EGFP或欺诈 dsRNA 处理的稳定 S2-Notch 单元之间进行聚合测定, 以检查反式绑定的效果。以 S2-Delta dsRNA 处理的纯 S2 单元格的聚合为背景控件. 欺诈与对照相比, KD 增强了 S2-Notch 和 S2-Delta 细胞的聚合。在与反相结合实验的同时, 对普通 S2 细胞和配体携带的 S2 细胞进行背景实验控制聚合是非常重要的。虽然果蝇S2 细胞不表达内源性切口受体和三角洲配体11, 但在 S2 细胞中报告的低锯齿水平的表达在13中。考虑到这一点, S2 细胞被用于细胞聚集测定作为背景控制;这些单元格没有显示任何聚合 (图 5B)。使用例手动计数聚合。为了确保总量量化的一致性, 10-20 µL 混合细胞的人口可以 pipetted 从至少三不同地区的每井计数。这些化验需要健康生长的 S2 细胞。考虑到 S2 细胞的 semi-adherent 性质, 在聚集过程中收集细胞和恒定的晃动时, 温和的移是很重要的。
以前, 我们和其他人已经使用了公认的, 可溶性配体受体结合检测的定量评估受体-配体结合在跨17,18,19,20. 由于聚合测定是半定量的, 因此还进行了可溶性配体结合物检测, 以检验在切口配体 反式结合的情况下, 欺诈性 KD 的作用。观察到的结果与从聚合分析中获得的数据的趋势相似8。这证明了用细胞聚合分析法对切口与反式配体结合的忠实评价。虽然集料的形成是受体-配体结合的读出, 它可以是复杂的, 如果表面表达的缺口受体或其配位受影响的给定的修饰符。这突出了细胞聚集分析的局限性, 因为它不能区分, 如果在聚合形成的变化是由于改变受体配体结合或改变表面表达。因此, 与聚合分析平行, 每个修饰剂对缺口和配体表面表达的影响需要在 S2 细胞中测试。在我们的研究中, 凹槽和三角洲的表面水平在 dsRNA 处理的 S2 细胞 8中不受影响。
为了通过添加cis-配体来检查对跨绑定的抑制作用, 在 S2-Delta 细胞和EGFP或 dsRNA 处理的 S2-Notch 和 #38;D elta 瞬态单元之间进行聚合分析.将这些单元格之间形成的聚合数与 S2-Delta 单元格和 S2-Notch瞬态单元格之间形成的聚合数进行比较。计算了骨料数量的相对减少, 并将其用作cis-三角洲抑制幅度的指标。对照实验表明,顺式-配体在相当宽的凹槽范围内显示了一个鲁棒和剂量依赖性的缺口/跨-三角洲结合的抑制作用 (1:1 到 1:0. 1) (图 5A)8.1:1 的比率导致了cis抑制的最强程度, 并降低了cis-三角洲减弱的cis抑制的相对水平。据此, 对这一比值范围进行了 KD 实验。提示, 在所有实验中, 切口受体的数量和转染 DNA 的总数量保持不变。这将确保聚合形成主要受cis-配体的活动的影响, 而不是由单元格的凹槽表达式的改变水平。
先前的研究表明, S2 细胞中的表达和顺式配体可以导致着聚合的形成14。要检查 S2-Notch 和 #38 中的类似着聚合是否;D elta瞬态或 S2-Notch 和 #38; 有锯齿的瞬态单元格可能会损害我们对cis配体研究中的聚合 quantifications 的解释,在 S2-Notch 和 #38;D elta瞬态和纯 S2 单元之间进行了控制聚合测定。这些实验中使用的 S2 细胞的数量与在cis-三角洲实验中使用的 S2-Delta 细胞相同。观察到了一些着聚合, 但量化结果表明, 与 S2-Notch 和 #38;D elta瞬态和 S2-Delta 细胞 (图 5B)。我们的结论是, 在顺式-配体测定中, 聚集体的数量所观察到的变化主要是由于顺式-配基在缺口与跨三角洲之间的结合上的抑制作用。
总之, 该协议中提出的聚合分析方法为对跨和cis-缺口受体及其配体的相互作用的半定量评估提供了一种简单明了的方法论。这些分析可用于检测基因或药理缺口通路修饰剂对切口配体结合的影响体外, 因此可以帮助阐明这些修饰符在凹槽中的作用机制。信号.

标准缺口信号是一个短程细胞的细胞通信机制, 需要物理接触相邻细胞, 以促进相互作用的缺口受体和他们的配体1。缺口受体与配体 (目前在信号接收细胞表面) 的相互作用 (目前在信号发送细胞的表面) 启动缺口信号, 称为反激活2。另一方面, 缺口与其配体在同一细胞中的相互作用导致切口通路的抑制, 被称为cis抑制3。跨-和cis-交互之间的平衡需要确保最佳配体依赖性缺口信号4。果蝇有一个缺口受体和两个配体 (三角洲和锯齿), 而不是哺乳动物, 其中有四缺口受体和五配体 [锯齿状 1 (JAG1), JAG2, 三角洲样 1 (DLL1), DLL3 和 DLL4]5。在这种简单的情况下,果蝇模型提供了易于解剖/研究通路修饰剂对缺口配体相互作用和随后的缺口信号的影响。在某些情况下在动物发展期间(包括翼发展在果蝇), 两个cis-和跨-相互作用参与获得适当的缺口信号和细胞命运16.在这些上下文中, 区分缺口路径修饰符对cis-与跨-缺口及其配体的相互作用的影响是很重要的。
我们的小组以前报告说, 添加一种叫做木糖的碳水化合物残渣到果蝇缺口在某些上下文中负调节缺口信号, 包括机翼开发7。损失的欺诈 (xylosylates 缺口的酶) 导致 "机翼静脉丢失" 表型 7。最近, 利用基因剂量试验和克隆分析表明, 欺诈的损失提高了三角洲介导的缺口. 要区分 "欺诈" 突变体中增强的缺口信号是否是由于减少了 cis抑制或增加了跨激活, 在幼虫翅形象盘中的缺口配体的异位表达研究已执行使用dpp-GAL4驱动程序。这些实验提供的证据表明, 在不影响凹槽cis的情况下, 在配体8的作用下, 该方法可调节反式--由 Delta 激活。然而, 反馈规则和内源性配体的作用可能使异位表达研究的解释复杂化1,6,9。
为解决此问题, 使用了果蝇S2 单元格10 , 它提供了一个简单的体外系统, 用于缺口配体相互作用研究11,12。S2 细胞不表达内源性缺口受体和三角洲配体11 , 表达低锯齿状的13, 这不影响缺口配体聚合实验8。因此, S2 细胞可以稳定或瞬时转染的缺口和/或个别配体 (三角洲或锯齿), 以产生细胞, 完全表达的缺口受体或其中一个配体, 或他们的组合。S2 细胞配体表达 S2 细胞的混合表现为由受体配体介导的异型团聚体的形成11,12,14。聚合形成的定量化提供了一种测量缺口与其配体之间的跨绑定的方法15 (图 1)。同样, S2 细胞可以 co-transfected 与缺口和三角洲或有锯齿配体 (即 cis-配体)。Cis-在这些凹槽表达 S2 细胞的配体中, 用反式配基废除凹槽的绑定, 并导致聚合形成减少8,12,14。由顺式-配体引起的集料形成的相对减少, 提供了一种在凹槽和反式配体 (图 2) 之间绑定的cis配体的抑制效果的测量。因此, 利用细胞聚集分析研究了 xylosylation 对反式和cis-缺口与配体之间相互作用的影响。
在这里, 我们提出了一个详细的细胞聚集试验的协议, 目的是评估的结合,反式配体和它的抑制, 由cis-配基使用果蝇S2 细胞。例如, 我们提供的数据使我们能够确定缺口 xylosylation 对缺口和跨-Delta8之间的绑定的影响。这些简单的分析提供了一个半定量评估缺口配体相互作用的体外和帮助确定的分子机制的基础上的体内的效果的缺口途径修饰。

<table><tbody><tr><td>BioWhittaker Schneider&amp;rsquo;s Drosophila medium, Modified</td><td>Lonza</td><td>04-351Q</td><td></td></tr><tr><td>HyClone Penicillin-Streptomycin 100X solution</td><td>GE Healthcare lifescience&amp;nbsp;</td><td>SV30010</td><td></td></tr><tr><td>CELLSTAR 6 well plate</td><td>Greiner Bio-One</td><td>657 160</td><td></td></tr><tr><td>CELLSTAR 24 well plate</td><td>Greiner Bio-One</td><td>662160</td><td></td></tr><tr><td>VWR mini shaker</td><td>Marshell Sceintific</td><td>12520-956</td><td></td></tr><tr><td>Hemocytometer</td><td>Fisher Sceintific&amp;nbsp;</td><td>267110</td><td></td></tr><tr><td>FuGENE HD Transfection Reagent</td><td>Promega</td><td>E2311</td><td></td></tr><tr><td>MEGAscrip T7 Transcription Kit</td><td>Ambion</td><td>AM1334</td><td></td></tr><tr><td>Quick-RNA MiniPrep (RNA purification Kit)</td><td>Zymo Research</td><td>R1054</td><td></td></tr><tr><td>VistaVision Inverted microscope</td><td>VWR</td><td></td><td></td></tr><tr><td>9MP USB2.0 Microscope Digital Camera + Advanced Software</td><td>AmScope</td><td>MU-900</td><td>Image acquisition using ToupView software</td></tr><tr><td>PureLink Quick Gel Extraction Kit</td><td>Invitrogen</td><td>K210012</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>GenDepot</td><td>F0600-050</td><td></td></tr><tr><td>Methotrexate</td><td>Sigma-Aldrich</td><td>A6770-10</td><td></td></tr><tr><td>Hygromycin B</td><td>Invitrogen</td><td>HY068-L6</td><td></td></tr><tr><td>Copper sulphate</td><td>Macron Fine Chemicals</td><td>4448-02</td><td></td></tr><tr><td>S2 cells</td><td>Invitrogen</td><td>R69007</td><td></td></tr><tr><td>S2-Serrate&lt;sup&gt;Tom &lt;/sup&gt;cells</td><td></td><td></td><td>Gift from R. Fleming (Fleming et al, Development, 2013)</td></tr><tr><td>S2-Delta cells</td><td>DGRC</td><td>152</td><td></td></tr><tr><td>S2-Notch cells</td><td>DGRC</td><td>154</td><td></td></tr><tr><td>pMT-Delta vector</td><td>DGRC</td><td>1021</td><td>Gift from S. Artavanis-Tsakonas</td></tr><tr><td>pMT-Serrate vector</td><td></td><td></td><td>Gift from Ken Irvine (Okajima et al, JBC, 2003)</td></tr><tr><td>pMT-Notch vector</td><td>DGRC</td><td>1022</td><td>Gift from S. Artavanis-Tsakonas</td></tr><tr><td>pAc5.1-EGFP</td><td></td><td></td><td>Gift from Hugo Bellen</td></tr><tr><td>TaqMan RNA-to-Ct 1-&lt;em&gt;Step&lt;/em&gt; Kit</td><td>Applied Biosystem</td><td>1611091</td><td></td></tr><tr><td>TaqMan Gene Expression Assay for CG9996 (Shams)</td><td>Applied Biosystem</td><td>Dm02144576_g1&amp;nbsp;</td><td>with FAM-MGB dye</td></tr><tr><td>TaqMan Gene Expression Assay for CG7939 (RpL32)</td><td>Applied Biosystem</td><td>Dm02151827_g1</td><td>with FAM-MGB dye</td></tr><tr><td>Applied Biosystems 7900HT Fast Real-Time PCR system</td><td>Applied Biosystem</td><td>4351405</td><td>96-well Block module</td></tr></tbody></table>

cell aggregation assays to evaluate the binding of the drosophila notch with trans-ligands and its inhibition by cis-ligands

缺口信号是一种进化保守的细胞-细胞通讯系统, 广泛用于动物发育和成人维护。缺口受体与相邻细胞配体的相互作用诱导信号通路激活 (反式激活), 而同一个细胞的配体相互作用抑制信号传导 (cis抑制)。在跨激活和cis抑制之间的适当平衡有助于在动物发育过程中的某些环境中建立最佳的缺口信号水平。由于凹槽及其配体在许多细胞类型中的重叠表达域和反馈机制的存在, 研究了给定的修饰修改对跨-与cis-缺口相互作用的影响它的配体在体内是困难的。在这里, 我们描述了一个协议, 使用果蝇S2 细胞在细胞聚集的分析, 以评估的影响, 敲下一个缺口通路修饰符的约束力, 每个配体在跨和cis。S2 细胞稳定或瞬时转染一个缺口表达载体与表达每个缺口配体 (S2-Delta 或 S2-Serrate) 的细胞混合。反式-受体与配体之间的结合导致异型细胞聚集物的形成, 并以每毫升和 #62 组成的集料数计算; 6 细胞。为了研究顺式-配体的抑制作用, S2 细胞 co-expressing 凹槽和各配基与 S2-Delta 或 S2-Serrate 细胞混合, 聚合数量按上述方法量化。由于顺式-配体的存在而导致的骨料数量相对减少, 提供了一种测量顺式-配基介导的抑制反式绑定的措施。这些简单的化验可以提供半定量的数据关于基因或药理操作对切口的结合的作用对它的配体, 并且能帮助破译基础的分子机制在体内作用这种对缺口信号的操控。

我们的在体内的观察表明, xylosyltransferase 基因的损失, 欺诈导致增益缺口信号由于增加三角洲介导的 跨-激活的缺口不影响cis抑制凹槽由配体8。为了测试这一概念,体外细胞聚合化验被执行。首先, S2 单元格中的欺诈表达式被使用 dsRNA 击倒. EGFP dsRNA 用作控件。用 real-time PCR 法检测了击倒 (KD) 的效率, 结果表明其大于 80%8。然后用聚合分析法检测了凹槽受体与反式-三角洲的结合。S2-Notch 和 S2-Delta 细胞之间的聚集在不同的时间点进行了混合。将普通 S2 细胞和 S2-Delta 细胞的聚集作为背景控制。图 3显示了混合后在不同时间点 (1、5、15和 30 min) 形成的聚合数的图形。在1和5分钟的团聚体数量上出现了急剧增加. 之后, 骨料的数量继续增加, 直到30分钟的速度减慢。因此, 在1、5和15分钟, 聚合体的形成被成像, 在5分钟的混合后, 骨料的数量被量化。
图 4A显示了在稳定的 S2-Notch 和 S2-Delta 细胞之间进行细胞聚合测定的代表性图像, 以评估欺诈 KD 对切口受体与 跨三角洲之间的结合作用。图像显示三时间点 (1、5和 15 min) 的聚合。对于背景控制, 纯 S2 单元格 (用 dsRNA 处理) 与 S2-Delta 单元格混合使用. EGFP dsRNA 处理的 S2-Notch 细胞形成了具有 S2-Delta 细胞的聚合体, 随时间增加。相比之下, "dsRNA" 处理后的 S2-Notch 单元格形成的聚合速度更快, 而聚合体的数量比控件的数目更大、更大 ( 图 4B)。这表明, 减少的欺诈级别将提高跨绑定的缺口, 并使用 Delta8。
为了研究是否会抑制cis-配体对凹槽和反式三角洲之间的约束作用, 我们首先确定了信号接收单元中的表达和三角形的衰减会降低细胞聚集由缺口和跨-三角洲介导。图 5A显示了在 S2-Delta 和 S2-Notch 之间形成的聚合数和 #38;D elta瞬态单元格的数量, 该值的cis-三角洲的数量不同。染等量的缺口和δ表达式构造成 S2 细胞, 大大减少了这些细胞和 S2-Delta 细胞之间形成的团聚体的数量。此外, 减少cis-增量的数量会导致聚合数的增加, 表明在这些分析中使用的缺口/cis-增量比率范围内, cis-三角洲可以抑制缺口和以剂量依赖性的方式反-三角洲。由于 S2-Notch 和 #38;D elta瞬态单元格同时表示凹槽和 Delta, 因此它们可能独立于 S2-Delta 细胞形成着聚合体。要检查在 S2-Notch 和 #38;D elta瞬态单元中着聚合的形成是否是cis抑制分析中的混杂因素, 如图 5A所示, 我们将这些细胞与 S2 细胞混合, 并量化料.图 5B显示了将 S2 单元与 S2-Notch 和 #38 混合后形成的聚合数;D elta瞬态单元格, 表示各种凹槽/cis-增量比率。与图 5A中所示的异型骨料相比, 着聚合体的数量相当少。我们得出的结论是, 这些聚合测试忠实地测量了cis-三角洲对缺口和跨-三角洲之间的绑定的影响。
接下来我们研究了欺诈 KD 对 cis-三角洲抑制能力的影响。为此, 检查了 S2-Delta 和 S2-Notch #38;D elta瞬态单元格之间的聚合。图 6A显示了在 S2-Delta 单元格和 S2-Notch瞬态(控制) 或 S2-Notch 和 #38;D elta瞬态单元格之间聚合的代表性图像。S2-Delta 和EGFP dsRNA 处理的 S2-Notch瞬态单元格形成聚合体, 随时间增加, 与 S2-Delta 和稳定 S2-Notch 细胞 (图 4) 之间的聚合类似。值得注意的是, 混合 S2-Delta 和 S2-Notch 和 #38;D elta瞬态单元 co-transfected 具有等量的凹槽和 Delta 表达质粒, 在EGFP kd 和欺诈的 kd 上形成较少的聚合体, 这意味着减少 欺诈的级别不影响cis-三角洲阻止缺口/跨-增量绑定的能力。为了更好地评估欺诈 KD 对 cis-三角洲活动的影响, 通过计算凹槽/cis三角洲范围内的相对聚集百分比来测量cis三角洲的抑制程度。比率, 如第3.4.2 节所述。相对聚合的量化显示了在EGFP和欺诈 dsRNA 处理 ( 图 6B) 上的聚合的类似抑制。同时, 这些观察强烈地表明, xylosylation 的丢失促进了跨绑定, 而不影响cis抑制8, 并提供了在欺诈中观察到的机翼静脉丢失表型的分子机制. 变种人。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56919/56919fig1.jpg"> 
图 1:跨绑定的单元聚合分析的示意图表示法。图表显示受体 (S2-Notch) 和配体 (S2-Delta 或 S2-Serrate) 表达 S2 细胞的 0.7 mM 丘索4。诱导两个 S2 细胞后, 混合, 这往往使聚合。这些聚合体 (#62; 6 细胞) 被成像和量化。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56919/56919fig1large.jpg" target="_blank">请点击这里查看更大版本的这个数字。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56919/56919fig2.jpg"> 
图 2: 用于评估Cis抑制的细胞聚集试验的示意图表示。S2 单元格瞬时转用pmt-缺口(S2-N瞬态)、 pmt 缺口和pmt-增量(S2-Notch 和 #38;D elta瞬态), 或pmt 缺口和pmt-锯齿(S2-Notch 和 #38;有锯齿的瞬态)。在诱导 0.7 mM 丘索4并与 S2-Delta 细胞混合后, 聚合体 (和 #62; 6 细胞) 被量化。由顺式-配体的抑制以相对聚集百分比的形式来衡量. cis-配体对缺口和跨-锯齿之间的结合作用可以通过将瞬时转染的细胞与 S2-SerrateTom细胞混合来确定 (未显示)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56919/56919fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56919/56919fig3.jpg"> 
图 3: 在不同时间点的 S2-Notch 和 S2 或 S2-Delta 细胞之间的聚合.图显示了在不同时间点 (1、5、15和 30 min) 之间在所指示的单元格类型之间每毫升的聚合数。误差线表示三复制的平均值 (SEM) 的标准误差。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56919/56919fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56919/56919fig4.jpg"> 
图 4: 通过缺口和跨-三角洲介导的单元聚合。() 图像在不同的时间点 (1、5和 15 min) 显示聚合.在欺诈 dsRNA 处理的纯 S2 单元格和 S2-Delta 单元格之间进行聚合作为背景控制. 在与 S2-Notch 细胞混合后, dsRNA 处理的 S2-Notch 细胞与 EGFP dsRNA 处理 (控制) S2-Delta 细胞相比, 聚合体的数量和大小都有增加。缩放条 = 100 µm. (B) 在聚合5分钟后, 对单元聚合数大于6单元的数量进行量化。误差线表示 SEM. * P和 #60; 0.01 (方差分析)。三复制与三独立重复执行。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56919/56919fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56919/56919fig5.jpg"> 
图 5: Cis-三角洲以剂量依赖性的方式抑制由缺口和跨三角洲介导的细胞聚集形成。(A) 图显示了 S2-Delta 细胞与 S2 细胞之间的聚合 (在聚合体/毫升数方面), 瞬时转染了不同的表达载体的比值, 并cis三角洲 (1:0、1:1、1:0. 5、1:0. 2 和 1:0. 1)。误差线表示 SEM. * P和 #60; 0.01 (单向方差分析)。三复制与三独立重复执行。请注意, 降低cis-增量的级别会导致聚合的增加, 这可能是由于cis抑制的减少。(B) 图显示着聚合 (按每毫升的聚合数计算) 在 S2 细胞瞬时转染不同缺口和三角洲的比率 (1:0, 1:1, 1:0. 5, 1:0. 2 和 1:0. 1), 并与纯 S2 细胞混合。误差线表示 SEM. 聚合体的数量比 S2-Delta 和 S2-Notch 和 #38 之间形成的异型骨料的数量要小得多,;D elta瞬态单元格。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56919/56919fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56919/56919fig6.jpg"> 
图 6: 欺诈的击倒不会影响 cis-三角洲的抑制活动。(A) 显示的是在 cis-三角洲-介导的细胞聚集抑制的影响的代表性图像, 通过缺口/跨-三角洲结合。图像在不同的时间点 (1、5和 15 min) 显示聚合。在欺诈 dsRNA 处理的纯 S2 单元格和 S2-Delta 单元格之间进行聚合作为背景控制. 与稳定转染的 S2-Notch 细胞相似, 格里斯 KD 增加了 S2-Delta 和 S2-Notch 瞬态单元格之间的聚合数, 与控件相比 (EGFP dsRNA 处理)。在1:1 的比率中, cis-三角洲的 Co 表达式导致了控制中的聚合数 (EGFP dsRNA 处理) 以及 dsRNA 处理的单元格的数量的相应减少. 缩放条 = 100 µm. (B) 图显示了 S2-Delta 细胞与 S2 细胞之间的相对聚集, 瞬时 co-transfected 的比值 (1:0、1:1、1:0. 5、1:0、2和 1:0) 的缺口和cis-三角洲。EGFP和 "欺诈" dsRNA 处理在每个缺口/ cis-增量比率中显示了相应的聚合减少。这里提供的数据是三独立实验的代表。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56919/56919fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands at JoVE.com

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

复杂的在体内系统, 使得很难区分的激活和抑制的缺口受体的跨-和cis配体, 分别。在这里, 我们提出了一个基于体外细胞聚合的方法, 用于定性和半定量评价的结合的果蝇缺口, 以跨配基与顺式配基。

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

cell-aggregation-assays-to-evaluate-binding-drosophila-notch-with

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

7.5K 次观看.  Baylor College of Medicine. 复杂的在体内系统, 使得很难区分的激活和抑制的缺口受体的跨-和cis配体, 分别。在这里, 我们提出了一个基于体外细胞聚合的方法, 用于定性和半定量评价的结合的果蝇缺口, 以跨配基与顺式配基。

视频: Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Integration of Bioinformatics Approaches and Experimental Validations to Understand the Role of Notch Signaling in Ovarian Cancer

Notch signaling is a highly conserved regulatory pathway involved in many cellular processes. Dysregulation of this signaling pathway often leads to interference with proper development and may even result in initiation or progression of cancers in certain cases. Because this pathway serves complex and versatile functions, it can be studied extensively through many different approaches. Of these, bioinformatics provides an undeniably cost-efficient, approachable, and user-friendly method of study. Bioinformatics is a useful way to extract smaller pieces of information from large-scale datasets. Through the implementation of various bioinformatics approaches, researchers can quickly, reliably, and efficiently interpret these large datasets, yielding insightful applications and scientific discoveries. Here, a protocol is presented for integration of bioinformatics approaches to investigate the role of Notch signaling in ovarian cancer. Furthermore, bioinformatics findings are validated through experimentation.

Bioinformatics is a useful way to process large-scale datasets. Through the implementation of bioinformatics approaches, researchers can quickly, reliably, and efficiently obtain insightful applications and scientific discoveries. This article demonstrates the utilization of bioinformatics in ovarian cancer research. It also successfully validates bioinformatics findings through experimentation.

生物信息学方法与实验验证的集成，了解诺奇信号在卵巢癌中的作用

Notch signaling is a highly conserved regulatory pathway involved in many cellular processes. Dysregulation of this signaling pathway often leads to ...

科研

癌症研究

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a G&#945;12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

Here we describe a contractility assay using Drosophila S2R+ cells. The application of an exogenous ligand, folded gastrulation (Fog), leads to the activation of the Fog signaling pathway and cellular contractility. This assay can be used to investigate the regulation of cellular contractility proteins in the Fog signaling pathway.

果蝇S2R+ 细胞折叠-肠信号通路检测非肌球蛋白 II . 收缩力的细胞基础研究

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted ...

发育生物学

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

现场剖析果蝇胚胎抗体染色筛选突变体集合的简化方法

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

生物学

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

检测<em&gt;原位</em&gt;蛋白质 - 蛋白质复合物的<em&gt;果蝇</em&gt;幼虫神经肌肉接头使用接近结扎分析

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

神经科学

通过抗体介导的实时成像增强蛋白质可视化

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

作者聚焦：揭示动物形态发生研究中机械和生化信号的动力学 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

通过异位细胞系统中的蛋白质聚合测量跨细胞相互作用

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

G蛋白偶联受体的基于荧光的钙动员含量表征

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

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

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

摘要

探索更多视频

生物信息学方法与实验验证的集成，了解诺奇信号在卵巢癌中的作用

果蝇S2R+ 细胞折叠-肠信号通路检测非肌球蛋白 II . 收缩力的细胞基础研究

现场剖析果蝇胚胎抗体染色筛选突变体集合的简化方法

检测

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

通过异位细胞系统中的蛋白质聚合测量跨细胞相互作用

G蛋白偶联受体的基于荧光的钙动员含量表征

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