该项目部分由美国国立卫生研究院国家癌症研究所的联邦资金资助，项目编号为 ZIA BC 010329。

注：该测定在 293FT 细胞中进行。来源于人胚胎肾细胞的特征明确且易于转染的上皮细胞系。这些细胞的单个汇合 10 cm 培养皿通常提供足够的细胞来接种 6 孔组织培养板的 20 孔。步骤 1-3 必须在生物安全柜中使用无菌技术进行。
1. 细胞接种（第 1 天）
注：在此步骤中，将细胞从组织培养皿中分离出来，计数并接种在 6 孔组织培养板中，以便在步骤 2 中进行转染（图 2）。
<ol><li>从 10 cm 培养皿中的细胞中吸出培养基。用 5 mL 磷酸盐缓冲盐水 （PBS） 洗涤细胞并吸出。</li>
	<li>加入 1 mL 胰蛋白酶-乙二胺四乙酸 （EDTA），并在 37 °C 下孵育 3-5 分钟，以将细胞从培养皿中分离出来。</li>
	<li>向细胞中加入 9 mL 完全 Dulbecco 改良的 eagle 培养基 （DMEM） 以中和胰蛋白酶，并反复上下移液以产生均匀的单细胞悬液。</li>
	<li>立即将 20 μL 细胞转移到 1.7 mL 试管中，并与 20 μL 台盼蓝染色剂混合。使用自动细胞计数仪（材料表）或血细胞计数器对细胞进行计数。</li>
	<li>在完全 DMEM 培养基中将细胞稀释至 2 x 105 个细胞/mL，并向 6 孔组织培养板的每个孔中加入 2 mL（4 x 105 个细胞/孔）。将细胞在 37 °C 和 10% CO2 下孵育过夜。 注：建议使用传代次数少于 20 次的 293FT 细胞。我们之前发现，使用传代次数更大的细胞会导致 BRET 比率降低和孔间信号变异性增加。</li>
</ol>2. 细胞转染（第 2 天）
注意：在这里，用 pCMV5-NanoLuc-CRAF 和 pCMV5-14-3-3ζ-Halo 表达构建体以及 pCDNA3.1 空载体转染细胞（图 2）。
<ol><li>转染前，将 N-BRET 质粒稀释至 5 ng/μL，将 pCDNA3.1 稀释至 100 ng/μL，并在一组无菌 1.7 mL 试管中编号。</li>
	<li>向每个试管中加入 100 μL 转染培养基（详见 材料表 ），以及 5 ng pCMV5-NanoLuc-CRAF、10 ng pCMV5-14-3-3ζ 和 200 ng PCDNA3.1。</li>
	<li>加入 2 μL 转染试剂（详见 材料表 ）并轻轻涡旋混合。在微量离心机中脉冲旋转试管，以确保所有液体都收集在试管底部，然后在 25 °C 左右孵育 15 分钟。</li>
	<li>向细胞中滴加转染复合物，并在 37 °C/10% CO2 下孵育 16-20 小时，以表达 Halo 和 Nano 标记的蛋白质。 注：添加空载体 （pCDNA3.1） 载体 DNA 对于实现 N-BRET 表达构建体的高转染效率至关重要。如前所述，未能添加空载体会导致 14-3-3ζ-Halo 和 Nano-CRAF 蛋白的表达水平降低，进而导致 BRET 比率较弱且不一致22。</li>
</ol>3. 细胞重新铺板（第 3 天）
注：在此步骤中，将细胞转移到 384 孔板和 Halo 618 配体（+配体; 材料表）或添加 DMSO （+vehicle） 以读取步骤 4 中的 BRET 发射。将剩余的细胞转移至新鲜的 6 孔培养板中，以便在步骤 5 中进行蛋白质印迹分析（图 2）。
<ol><li>收集以下材料并按照以下说明准备工作区域。<ol><li>使用 37 °C 水浴，预热胰蛋白酶-EDTA，以及无血清检测培养基和 10% FBS 补充的检测培养基（有关详细信息 ，请参阅材料表 ）。</li>
		<li>根据要测量的样品数量，预先标记三组无菌 1.7 mL 试管（套装 1-3）和一组无菌 15 mL 锥形底管。在水平转子离心机上配备 15 mL 试管插件，并预冷至 4 °C。</li>
		<li>将以下物品放入组织培养罩中：试剂储液槽、384 孔组织培养板、6 孔组织培养板、多通道移液器和吸头、细胞计数玻片/腔室和台盼蓝染色剂（材料表），以及 1.7 mL 试管组 1-3。</li>
	</ol></li>
	<li>如下所述收获并计数细胞。<ol><li>从 6 孔板中吸出培养基，并向细胞中加入 250 μL 胰蛋白酶-EDTA。在 37 °C 下孵育 6 孔板，直到细胞开始分离（3-5 分钟）。</li>
		<li>向每个孔中加入 1 mL 补充 10% FBS 的检测培养基以中和胰蛋白酶，并用力上下移液以产生单细胞悬液。</li>
		<li>将 1 mL 细胞悬液转移到预先标记的 15 mL 试管中。向每个 15 mL 试管中再添加 1 mL 补充 10% FBS 的检测培养基。</li>
		<li>将试管倒置 5 次以混合，并立即将 20 μL 细胞悬液转移到试管组 1 中。</li>
		<li>在预冷的水平吊篮离心机中以 ~250 x g 的速度离心 5 分钟。在离心步骤中，将 20 μL 台盼蓝细胞染色剂与管组 1 中的细胞混合，然后使用自动细胞计数仪或血细胞计数器对细胞进行计数。典型产量为 6-8 x 105 个细胞/mL。</li>
		<li>从离心机中取出 15 mL 试管，并从细胞沉淀中吸出培养基。在无血清检测培养基中将细胞沉淀重悬至 2 x 105 个细胞/mL，并用力移液以生成单细胞悬液。 注：使用无血清检测培养基用于静止正常细胞信号通路。</li>
	</ol></li>
	<li>如下所述，在 384 孔板和 6 孔板中重新铺板。<ol><li>将 15 mL 试管倒置数次以确保细胞悬液均匀，并将 500 μL 转移至 1.7 mL 试管 2 组和组 3 中。</li>
		<li>将 0.5 μL DMSO（+载体）添加到组 2 中，将 0.5 μL Halo 618 配体（+配体）添加到管组 3 中，然后移液混合。</li>
		<li>将 +载体和 +配体细胞悬液转移到试剂储液槽的单独孔中。使用多通道移液器（材料表），将 40 μL + 载体细胞悬液从试剂储液槽转移到 384 孔培养板的四份孔中。对 +配体细胞悬液重复此步骤。</li>
		<li>将剩余细胞转移到新鲜的 6 孔培养板中。将 384 孔板和 6 孔板在 37 °C 和 5% CO2 下孵育过夜。</li>
	</ol></li>
</ol>4. 读取 BRET 排放（第 4 天）
注：在此步骤中，将纳米荧光素酶底物（详见 材料表 ）添加到 384 孔培养板中的细胞中，并读取 N-BRET 受体 （618 nm） 和供体 （460 nm） 发射（图 2）。然后计算校正后的 BRET 比率。
<ol><li>在 37 °C 水浴中预热无血清检测培养基，并在 25°C 下解冻纳米荧光素酶底物。 在无血清检测培养基中以 1：100 的比例稀释纳米荧光素酶底物，然后转移到试剂储液槽中。准备足够的这种混合物，向 384 孔板的每个孔中加入 10 μL，外加 10%-15% 的额外体积。</li>
	<li>使用多通道移液器（材料表），将 10 μL 底物混合物转移到 384 孔培养板中含有细胞的每个孔中。轻轻旋转板 1 分钟，手动或使用轨道振荡器。</li>
	<li>将 384 孔板插入多功能读板器中，并记录所有包含细胞的孔的 460 nm 和 618 nm 发射。 注意：对于用于此步骤的多功能读板器（有关详细信息，请参阅 材料表 ），使用了 6.5 mm 的读取高度和 0.1 s 的读取时间测量，但是如果使用其他读板器，则可能需要进一步优化。</li>
	<li>使用以下等式分别计算 +载体和 +配体的原始 BRET 比率，以 milliBRET 单位 （mBU） 为单位： <img alt="Equation 1" src="/files/ftp_upload/66436/66436eq01.jpg" style="margin: auto;"></li>
	<li>通过从 +配体的 BRET 比率中减去 +载体 BRET 比率来计算校正的 BRET 比率（补充表 1）。 注：当比较多个实验的结果时，校正后的 BRET 比率可以合并并标准化为野生型 （WT） N-BRET 对的平均值，该对由 Nano-CRAFWT 和 14-3-3ζWT-Halo 组成（补充表 1）。</li>
</ol>5. 确认蛋白表达水平（第 4 天和第 5 天）
注意：在此步骤中，裂解 6 孔板中的细胞，并使用对 Halo 和 Nano 标签具有特异性的抗体通过蛋白质印迹分析确定 Nano-CRAF 和 14-3-3ζ-Halo 蛋白的蛋白质表达水平。（图 2）。
<ol><li>向 NP40 裂解缓冲液中加入蛋白酶和磷酸酶抑制剂（材料表），每个样品可使用 200 μL 裂解缓冲液。</li>
	<li>从含有细胞的 6 孔板中吸出培养基。用 1 mL 冷 PBS 洗涤细胞一次并吸出。</li>
	<li>向每个孔中加入 125 μL NP40 裂解缓冲液，并在 4 °C 下在摇动平台上孵育 6 孔板 15 分钟以裂解细胞。</li>
	<li>将裂解物转移至冰上的 1.7 mL 试管中，并在 4 °C 下以 20,000 x g 离心 10 分钟。 将澄清的裂解物放回冰上，并使用市售的测定法测定蛋白质浓度，例如 Bradford 或二辛可宁酸 （BCA） 测定法。</li>
	<li>通过将适当体积的裂解物和 NP40 裂解缓冲液转移到新鲜的 1.7 mL 试管中，使所有样品中的蛋白质浓度标准化，总体积为 100 μL。</li>
	<li>将 5x 蛋白质样品缓冲液（材料表）煮沸 1 分钟，并向每个样品中加入 25 μL。将样品煮沸 5-6 分钟，然后短暂脉冲离心试管，以确保所有样品都聚集在试管底部。</li>
	<li>将 25 μL 样品上样到重复蛋白质凝胶（凝胶 1 和凝胶 2）的每个孔中，然后使用标准蛋白质印迹程序将蛋白质转移到硝酸纤维素或 PVDF 膜上，如前所述22。</li>
	<li>在 25 °C 下用 3% BSA-PBS 封闭膜 30 分钟，然后用 Halo 抗体（1：1,000 稀释;凝胶 1）和纳米荧光素酶或 CRAF 抗体（1：500 稀释;凝胶 2）溶于 tris 缓冲盐水中，补充有 0.2% Tween-20 （TBST; 材料表）。</li>
	<li>在 10 mL TBST 中洗涤膜 1 次，每次 5 分钟，然后在室温下与在 TBST 中以 1：10,000 稀释的抗小鼠 HRP 二抗孵育 1 小时。</li>
	<li>在摇动平台上用 10 mL TBST 在 25 °C 下洗涤膜 3 次，每次 5 分钟。去除 TBST 并使用 ECL 试剂和 X 射线胶片处理器或其他合适的成像系统观察蛋白质条带。</li>
</ol>

使用基于 BRET 的方法在活细胞中进行 CRAF-14-3-3 相互作用分析

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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=14-3-3+zeta+negatively+regulates+raf-1+activity+by+interactions+with+the+Raf-1+cysteine-rich+domain.">14-3-3 zeta negatively regulates raf-1 activity by interactions with the Raf-1 cysteine-rich domain.</a> J Biol Chem. 272, 20990-20993 (1997).</li><li>Machleidt, 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=NanoBRET--A+novel+BRET+platform+for+the+analysis+of+protein-protein+interactions.">NanoBRET--A novel BRET platform for the analysis of protein-protein interactions.</a> ACS Chem Biol. 10, 1797-1804 (2015).</li><li>Hekman, 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=Dynamic+changes+in+C-Raf+phosphorylation+and+14-3-3+protein+binding+in+response+to+growth+factor+stimulation:+differential+roles+of+14-3-3+protein+binding+sites.">Dynamic changes in C-Raf phosphorylation and 14-3-3 protein binding in response to growth factor stimulation: differential roles of 14-3-3 protein binding sites.</a> J Biol Chem. 279, 14074-14086 (2004).</li><li>Hatzivassiliou, G., 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=RAF+inhibitors+prime+wild-type+RAF+to+activate+the+MAPK+pathway+and+enhance+growth.">RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.</a> Nature. 464, 431-435 (2010).</li><li>Bondzi, C., Grant, S., Krystal, G. W. <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+assay+for+the+measurement+of+Raf-1+kinase+activity.">A novel assay for the measurement of Raf-1 kinase activity.</a> Oncogene. 19, 5030-5033 (2000).</li><li>Spencer-Smith, R., 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=Inhibition+of+RAS+function+through+targeting+an+allosteric+regulatory+site.">Inhibition of RAS function through targeting an allosteric regulatory site.</a> Nat Chem Biol. 13 (1), 62-68 (2016).</li><li>Roy, S., 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=14-3-3+facilitates+Ras-dependent+Raf-1+activation+in+vitro+and+in+vivo.">14-3-3 facilitates Ras-dependent Raf-1 activation in vitro and in vivo.</a> Mol Cell Biol. 18, 3947-3955 (1998).</li><li>Durrant, D. 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=Development+of+a+high-throughput+NanoBRET+screening+platform+to+identify+modulators+of+the+RAS/RAF+interaction.">Development of a high-throughput NanoBRET screening platform to identify modulators of the RAS/RAF interaction.</a> Mol Cancer Ther. 20, 1743-1754 (2021).</li></ol>

没什么可透露的。

先前的研究表明，14-3-3 蛋白在 RAF 激酶的激活和抑制中起关键作用。了解这些结合事件如何被调节以及调节这些相互作用对 RAF 信号转导和 RAF 驱动的肿瘤发生的影响可能会发现针对 CRAF 功能的新治疗脆弱性。然而，Raf 激活周期受到大量相关蛋白、翻译后修饰和亚细胞定位变化的支持8，因此，为了完全概括这些条件，必须在活细胞条件下测量 RAF 与其调节因子的相互作用。该协?...

RAF 激酶 （ARAF、BRAF 和 CRAF） 是 RAS GTP 酶的直接效应子，也是促增殖/促存活 RAF-MEK-ERK 激酶级联反应的起始成员。最近的研究表明，CRAF 表达在几种 KRAS 驱动的癌症的肿瘤发生中起关键作用，包括非小细胞肺癌和胰腺导管腺癌 1,2,3,4,5。此外，种系 CRAF 突变会导致一种特别严重的 RASopathy，即 Noonan 综合征 6,7。了解 CRAF 调节对于开发针对其在细胞中的功能的成功治疗方法至关重要。
所有 RAF 激酶均可分为两个功能结构域，即 C 端催化 （CAT） 结构域和 N 端调节 （REG） 结构域，用于控制其活性（图 1A）8。REG 结构域包括 RAS 结合结构域 （RBD）、富含半胱氨酸的结构域 （CRD） 和富含丝氨酸/苏氨酸的区域 （S/T 富集）。值得注意的是，富含 S/T 的区域包含 N' 位点，该位点以磷酸化依赖性方式与 14-3-3 结合（CRAF 中的 S259;图 1A）8. CAT 结构域包括激酶结构域，以及第二个高亲和力 14-3-3 对接位点，称为 C' 位点（CRAF 中的 S621;图 1A）8. 二聚体 14-3-3 蛋白与 N' 和 C' 位点的差异结合以及 CRD 在 RAF 激活和抑制中起关键作用 9,10,11,12,13。在正常信号转导条件下，RAF 激活是通过 RAS 募集到质膜而启动的，使其能够形成活性二聚体，其中 BRAF-CRAF 异二聚体是主要的活性形式14,15。使用 BRAF 和 CRAF 的生化测定以及二聚体 BRAF 的低温电子显微镜 （Cryo-EM） 结构表明，14-3-3 二聚体通过同时结合两个 RAF 原聚体的 C' 位点来稳定活性 RAF 二聚体（图 1B）9,13,16,17。相反，研究表明，在静止条件下，RAF 采用胞质性、自抑制确认，其中 REG 结构域与 CAT 结构域结合并抑制其活性 12,18,19,20。这种闭合状态通过与 REG 结构域中的 CRD 和 N' 位点以及 CAT 结构域中的 C' 位点结合的 14-3-3 二聚体稳定（图 1B）10,13,21。在 BRAF 中，该模型得到了自体抑制 BRAF 单体的最新冷冻电镜结构以及我们之前的生化研究的支持 10,12,13,21,22。然而，虽然 14-3-3 在 CRAF 调节中起抑制作用23，但类似 BRAF 的自身抑制状态在 CRAF 调节中的作用可能较小12;因此需要进一步的研究来阐明 14-3-3 蛋白调节 CRAF 活性的机制。14-3-3 介导的 RAF 激酶调节需要大量的 RAF 磷酸化和去磷酸化事件、与各种调节蛋白的结合以及与质膜的相互作用8。因此，在生理相关条件下和存在完整脂质双层的情况下测量 14-3-3-RAF 相互作用至关重要。
为了解决这个问题，利用 NanoBRET（以下简称 N-BRET;试剂盒详情见材料表）技术开发了一种基于邻近度的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用（图 1C）。这种基于 BRET 的系统可测量两种目标蛋白 （POI） 的相互作用，其中一种蛋白质用纳米荧光素酶 （Nano） 供体标记，另一种用 Halo 标签标记，以便用 Halo618 能量受体配体标记22,24。目标蛋白质的相互作用导致供体到受体的能量转移，进而产生 BRET 信号（图 1C）。与传统 BRET 相比，极亮的纳米供体蛋白（发射 （em） 460 nm）和 Halo618 配体 （em 618 nm） 可提供更高的光谱分离和灵敏度，使其成为研究较弱相互作用和检测结合细微变化的理想平台24。事实上，我们之前开发了一种基于 N-BRET 的测定法，用于测量 RAF REG 和 CAT 结构域的自抑制相互作用，这对于表征 BRAF CRD 中的一组 RASopathy 突变至关重要，并证明了该结构域对于维持自身抑制和防止组成型 BRAF 激活的至关重要12。
此处描述的测定法测量与 N 端纳米标签 （Nano-CRAF） 融合的 CRAF 和与 C 端 Halo 标签融合的 14-3-3 的 zeta 亚型（14-3-3ζ-Halo; 图 1C）。我们表明，Nano-CRAF 与 14-3-3ζ-Halo 的相互作用会产生强大的 BRET 信号，该信号反过来又可以被阻止 14-3-3 与 N' 位点 （S259A） 和/或 C' 位点 （S621A） 结合的突变破坏。以下方案提供了执行、优化和排除此分析的详细步骤。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HaloTag&#174; mouse monoclonal antibody</td>
			<td>Promega</td>
			<td>G9211&#160;</td>
			<td>Antibody for detecting HaloTag tagged&#160; proteins by immunoblot</td>
		</tr>
		<tr>
			<td>NanoLuc&#174; mouse monoclonal antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB10026</td>
			<td>Antibody for detecting Nano-tagged proteins by immunoblot</td>
		</tr>
		<tr>
			<td>CRAF mouse monoclonal antibody (E10)&#160;</td>
			<td>Santa Crus Biotechnology</td>
			<td>sc-7267</td>
			<td>Antibody directly detecting CRAF proteins by immunoblot</td>
		</tr>
		<tr>
			<td>ECL anti-mouse HRP secondary antibody</td>
			<td>Amersham</td>
			<td>NA931-1ML&#160;</td>
			<td>Secondary HRP conjugated mouse antibody (from sheep)</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X-tremeGENE&#8482; 9</td>
			<td>Roche/Sigma</td>
			<td>6365809001</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoBRET&#8482; kit&#160;</td>
			<td>Promega</td>
			<td>N1661&#160;</td>
			<td>NanoBRET kit containing Halo 618 ligand and NanoGlo (nanoluciferase) substrate&#160;</td>
		</tr>
		<tr>
			<td>DPBS, without Ca++ and Mg++&#160;</td>
			<td>Quality Biologicals&#160;</td>
			<td>114-057-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red&#160;</td>
			<td>Life Technologies</td>
			<td>25300120</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM cell culture media</td>
			<td>Life Technologies</td>
			<td>11995073</td>
			<td>High glucose, L-glutamine, phenol red, sodium&#160; pyruvate; without HEPES, suppliment media with 10% FBS, 2 mM L-glutamine and 100U penicillin-streptomycin</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)&#160;&#160;</td>
			<td>Life Technologies</td>
			<td>25030164</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)&#160;</td>
			<td>Life Technologies&#160;&#160;</td>
			<td>15140163</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM&#8482; I reduced serum media&#160;</td>
			<td>Gibco</td>
			<td>31985062</td>
			<td>For cell transfection&#160;</td>
		</tr>
		<tr>
			<td>Opti-MEM reduced serum media, no phenol red</td>
			<td>Gibco</td>
			<td>11058021</td>
			<td>For replating cells on Day 3. Supplement with 2 mM L-glutamine and 100U penicillin-streptomycin, along with 10% FBS (where indicated).&#160;</td>
		</tr>
		<tr>
			<td>Invitrogen Trypan Blue Stain&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>T10282&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>NP40 lysis buffer&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>20 mM Tris (pH 8.0), 137mM NaCl, 10% glycerol,&#160; NP40 alternative (Milipore, Cat# 492016). Store at 4 degrees C.. Add the following protease and phosphatase immediately prior to use: 20 &#181;M leupeptin, 0.5 mM sodium orthovanidate, 0.15 U/mL, 1mM PMSF.</td>
		</tr>
		<tr>
			<td>5x gel sample buffer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>240 mM Tris (pH 8.0), 9.5% SDS, 30% glycerol, 500mM DTT, 3mM bromophenol blue. Store at -20 degrees C.&#160;</td>
		</tr>
		<tr>
			<td>Cell lines&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>293FT cells (human)</td>
			<td>Thermo Scientific&#160;</td>
			<td>R70007&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA vectors&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV5-Nano-CRAF WT and mutant&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV5-14-3-3&#950;-Halo&#160;&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EnVision 2104 Multimode Plate Reader</td>
			<td>PerkinElmer 2104</td>
			<td>2104-0010&#160;</td>
			<td>600LP NanoBRET &#38; M460/50 nm NanoBRET emmisions filters,&#160; Luminescence 404 mirror, 6.5 mm measurement height and 0.1 s measurement time</td>
		</tr>
		<tr>
			<td>Invitrogen Countess&#8482; II Automated Cell Counter&#160;</td>
			<td>Thermo Scientific</td>
			<td>AMQAX1000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoFisher E1-ClipTip&#8482;&#160; Multichannel&#160; Pipettor&#160;</td>
			<td>Thermo Scientific&#160;&#160;</td>
			<td>4672070</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism (version 10.0.3)&#160;</td>
			<td>GraphPad&#160;</td>
			<td>www.graphpad.com</td>
			<td></td>
		</tr>
		<tr>
			<td>Other&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoFisher ClipTip Multichannel pipette tips&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>94410153</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent Reservoir, 25 mL Divided, Sterile&#160;</td>
			<td>Thomas Scientific</td>
			<td>1228K16</td>
			<td></td>
		</tr>
		<tr>
			<td>Perkin Elmer 384-well CulturPlate&#8482;</td>
			<td>PerkinElmer</td>
			<td>6007680</td>
			<td>White, polystyrene, tissue culture treated&#160;</td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Thermo Scientific&#160;</td>
			<td>C10228</td>
			<td></td>
		</tr>
	</tbody>
</table>

bioluminescence resonance energy transfer (bret)-based assay for measuring interactions of craf with 14-3-3 proteins in live cells

CRAF 是 RAS GTP 酶的主要效应子，在几种 KRAS 驱动的癌症的肿瘤发生中起关键作用。此外，CRAF 是种系突变的热点，种系突变被证明会导致发育性 RASopathy、Noonan 综合征。所有 RAF 激酶都包含 14-3-3 调节蛋白的多个磷酸化依赖性结合位点。14-3-3 与这些位点的差异结合在信号转导条件下质膜上活性 RAF 二聚体的形成和在静止条件下维持 RAF 自抑制中起着重要作用。了解这些相互作用如何被调节以及如何调节它们对于确定靶向 RAF 功能的新治疗方法至关重要。在这里，我描述了一种基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用。具体来说，该测定法测量了与纳米荧光素酶供体融合的 CRAF 和与 Halo 标签受体融合的 14-3-3 的相互作用，其中 RAF 和 14-3-3 的相互作用导致供体到受体的能量转移和 BRET 信号的产生。该方案进一步表明，该信号可以被显示阻止 14-3-3 与其每个高亲和力 RAF 停靠位点结合的突变破坏。该实验步骤描述了接种、转染和重新铺板细胞的程序，以及读取 BRET 发射、进行数据分析和确认蛋白质表达水平的详细说明。此外，还提供了示例分析结果以及优化和故障排除步骤。

RAF kinases (ARAF, BRAF, and CRAF) are the direct effectors of RAS GTPases and the initiating members of the pro-proliferative/pro-survival RAF-MEK-ERK kinase cascade. Recent studies have shown that CRAF expression plays a key role in the tumorigenesis of several KRAS-driven cancers, including non-small cell lung cancer and pancreatic ductal adenocarcinoma1,2,3,4,5. Moreover, germline CRAF mutations cause a particularly severe form of the RASopathy, Noonan syndrome6,7. Understanding CRAF regulation is critical for developing successful therapeutic approaches that target its function in cells.
All RAF kinases can be divided into two functional domains, a C-terminal catalytic (CAT) domain and an N-terminal regulatory (REG) domain, that controls its activity (Figure 1A)8. The REG domain encompasses the RAS binding domain (RBD), the cysteine-rich domain (CRD), and a serine/threonine-rich region (S/T-rich). Notably, the S/T-rich region contains the N&#39; site, which binds to 14-3-3 in a phosphorylation-dependent manner (S259 in CRAF; Figure 1A)8. The CAT domain encompasses the kinase domain, along with a second high-affinity 14-3-3 docking site, referred to as the C&#39; site (S621 in CRAF; Figure 1A)8. The differential binding of dimeric 14-3-3 proteins to the N&#39; and C&#39; sites, along with the CRD, plays critical roles in both RAF activation and inhibition9,10,11,12,13. Under normal signaling conditions, RAF activation is initiated by its recruitment to the plasma membrane by RAS, allowing it to form active dimers, of which the BRAF-CRAF heterodimer is the predominant active form14,15. Biochemical assays with BRAF and CRAF, along with cryogenic electron microscopy (Cryo-EM) structures of dimeric BRAF, indicate that a 14-3-3 dimer stabilizes active RAF dimers by binding simultaneously to the C&#39; site of both RAF protomers (Figure 1B)9,13,16,17. Conversely, studies have shown that under quiescent conditions, RAF adopts a cytosolic, autoinhibited confirmation, where the REG domain binds to the CAT domain and inhibits its activity12,18,19,20. This closed state is stabilized by a 14-3-3 dimer bound to the CRD and N&#39; site in the REG domain and to the C&#39; site in the CAT domain (Figure 1B)10,13,21. In BRAF, this model is supported by recent Cryo-EM structures of autoinhibited BRAF monomers and by our previous biochemical studies10,12,13,21,22. However, while 14-3-3 is shown to play an inhibitory role in CRAF regulation23, a BRAF-like autoinhibited state may play a lesser role in CRAF regulation12; therefore further studies are required to clarify the mechanisms by which 14-3-3 proteins regulate CRAF activity. The 14-3-3-mediated regulation of RAF kinases requires a plethora of RAF phosphorylation and de-phosphorylation events, the binding to various regulatory proteins, and interactions with the plasma membrane8. Therefore, it is critical that 14-3-3-RAF interactions are measured under physiologically relevant conditions and in the presence of an intact lipid bilayer.
To address this issue, NanoBRET (from here on referred to as N-BRET; see Table of Materials for kit details) technology was utilized to develop a proximity-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells (Figure 1C). This BRET-based system measures the interactions of two proteins of interest (POI), where one protein is tagged with a nanoluciferase (Nano) donor and the other with a Halo tag, for labeling with the Halo618 energy acceptor ligand22,24. Interaction of the proteins of interest results in donor to acceptor energy transfer, which in turn generates the BRET signal (Figure 1C). The extremely bright Nano donor protein (emission (em) 460 nm) and the Halo618 ligand (em 618 nm) provide greater spectral separation and sensitivity over conventional BRET, making it an ideal platform for studying weaker interactions and detecting subtle changes in binding24. Indeed, we previously developed a N-BRET-based assay for measuring the autoinhibitory interactions of the RAF REG and CAT domains, which was essential for the characterization of a panel of RASopathy mutations in the BRAF CRD and demonstrated the critical importance of this domain for maintaining autoinhibition and preventing constitutive BRAF activation12.
The assay described here measures the interactions of CRAF, fused to an N-terminal Nano tag (Nano-CRAF), and the zeta isoform of 14-3-3 fused to C-terminal Halo tag (14-3-3&#950;-Halo; Figure 1C). We show that the interactions of Nano-CRAF with 14-3-3&#950;-Halo generates a robust BRET signal, which can in turn be disrupted by mutations which prevent 14-3-3 binding to the N&#39; site (S259A) and/or the C&#39; site (S621A). The following protocol provides detailed steps for performing, optimizing, and troubleshooting this assay.

作者聚焦：整合基于 BRET 的检测和罕见突变分析以破译活细胞中的 RAF 激酶调控

基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

The primary focus of our research is to uncover new vulnerabilities in the Ras-MAPK kinase pathway through the study of rare disease associated mutations within. We recently developed another BRET-based assay for measuring the auto inhibitory activities of RAF kinases in live cells. Importantly, this assay was essential for characterizing a group of rare germline mutations in the BRAF cysteine rich domain, which demonstrated the critical importance of this domain for maintaining BRAF auto inhibition and for preventing constitutive BRAF biological activity.This assay measures the interactions of 1433 proteins with the RAF kinase CRAF, and these interactions play essential roles in mediating RAF function in both development and disease. In addition, this assay combined with previously established assays for measuring RAF auto inhibition and the RAF-RAF interaction form the basis of a toolkit for dissecting the regulatory mechanisms of RAF kinases in live cells.

当按照本协议中的描述进行时（图 2），Nano-CRAFWT 和 14-3-3ζ-Halo 的相互作用应产生 50-60 mBU 的校正 BRET 比率（图 3A;补充表 1）。CRAF 包含两个磷酸化依赖性 14-3-3 停靠位点，即 N' 位点和 C' 位点（图 1）8。因此，减少 CRAF：14-3-3ζ 结合的适当对照包括 S259A 和 S621A，它们分别阻止磷酸化，进而阻?...

Read this Scientific Article Protocol about Author Spotlight: Integrating BRET-Based Assays and Rare Mutation Analysis to Decipher RAF Kinase Regulation in Live Cells at JoVE.com

该方案描述了一种基于 BRET 的测定法，用于测量 CRAF 激酶与活细胞中 14-3-3 蛋白的相互作用。该协议概述了准备细胞、读取 BRET 发射和数据分析的步骤。还提供了一个示例结果，其中确定了适当的对照和分析优化的故障排除。

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot ...

我们研究的主要重点是通过研究罕见病相关突变来发现 Ras-MAPK 激酶通路中的新脆弱性。我们最近开发了另一种基于 BRET 的测定法，用于测量活细胞中 RAF 激酶的自身抑制活性。重要的是，该测定对于表征 BRAF 富含半胱氨酸结构域中的一组罕见种系突变至关重要，这证明了该结构域对于维持 BRAF 自身抑制和防止组成型 BRAF 生物活性至关重要。该测定法测量 1433 种蛋白质与 RAF 激酶 CRAF 的相互作用，这些相互作用在介导发育和疾病中的 RAF 功能中起着重要作用。此外，该测定与先前建立的用于测量 RAF 自身抑制和 RAF-RAF 相互作用的测定相结合，构成了剖析活细胞中 RAF 激酶调节机制的工具包的基础。

bioluminescence-resonance-energy-transfer-bret-based-assay-for

Research

JoVE Journal

Biochemistry

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells

639 次观看.  Medical University of South Carolina. 我们研究的主要重点是通过研究罕见病相关突变来发现 Ras-MAPK 激酶通路中的新脆弱性。我们最近开发了另一种基于 BRET 的测定法，用于测量活细胞中 RAF 激酶的自身抑制活性。重要的是，该测定对于表征 BRAF 富含半胱氨酸结构域中的一组罕见种系突变至关重要，这证明了该结构域对于维持 BRAF 自身抑制和防止组成型 BRAF 生物活性至关重要。该测定法测量 1433 种蛋白质与 ...

视频: 作者聚焦：整合基于 BRET 的检测和罕见突变分析以破译活细胞中的 RAF 激酶调控

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

This protocol describes a BRET-based assay for measuring the interactions of the CRAF kinase with 14-3-3 proteins in live cells. The protocol outlines steps for preparing the cells, reading BRET emissions, and data analysis. An example result with identification of appropriate controls and troubleshooting for assay optimization is also presented.

科研

生物化学

Transfection of 293FT Cells with pCMV5-NanoLuc-CRAF and pCMV5-14-3-3&#950;-Halo Expression Constructs

To begin, take 293FT cells cultured in a 10 centimeter dish, and aspirate the media. Wash cells with five milliliters of PBS. Then add one milliliter of trypsin EDTA, and incubate for 3-5 minutes at 37 degrees Celsius to detach cells from the dish.After incubation, add nine milliliters of complete DMEM to the cells to neutralize the trypsin. Pipette up and down repeatedly to generate a homogenous single cell suspension, and transfer the cells to a 15 milliliter tube. Immediately transfer 20 microliters of cells to a 1.7 milliliter tube, and mix with 20 microliters of trypan blue stain.Count cells using either an automated cell counter or a hemocytometer. Transfer the appropriate volume of cells to a 50 milliliter tube, and dilute in complete DMEM. Add two milliliters of cell suspension to each well of a six-well plate.Incubate cells at 37 degrees Celsius and 10%carbon dioxide overnight. Prior to transfection, dilute nanoluciferase CRAF and 1433 zeta halo plasmids, along with a pCDNA3 empty vector in TE buffer. Label a set of sterile 1.7 milliliter tubes.Add 100 microliters of transfection media to each tube, along with the expression constructs. Now add two microliters of transfection reagent, and vortex gently to mix. Pulse spin the tubes briefly in a microcentrifuge, and then incubate the tubes at around 25 degrees Celsius for 15 minutes to allow transfection complexes to form.Afterwards, add transfection complexes to cells dropwise, and incubate at 37 degrees Celsius for 16-20 hours to express the halo and nanotagged proteins.

用 pCMV5-NanoLuc-CRAF 和 pCMV5-14-3-3ζ-Halo 表达构建体转染 293FT 细胞

Preparing 293FT Cells Transfected with pCMV5-NanoLuc-CRAF and pCMV5-14-3-3&#950;-Halo for BRET Emission Assay

To begin, label three sets of sterile 1.7-milliliter tubes and one set of sterile 15-milliliter conical bottom tubes. Prepare a swing bucket centrifuge with 15-milliliter tube inserts and pre-cool to four degrees Celsius. Take the six-well cell culture plate with transfected 293FT cells.Aspirate the media from the wells. Add 250 microliters of trypsin-EDTA and incubate at 37 degrees until cells begin to detach. Then add one milliliter of 10%FBS supplemented assay media to each well and pipette vigorously to create a single-cell suspension.Transfer one milliliter of cell suspension to pre-labeled 15-milliliter tubes. Add one milliliter of 10%FBS supplemented assay media to each of these tubes and invert five times to mix. Then immediately transfer 20 microliters of cell suspension to tube set one.Now centrifuge the 15 milliliter tubes for five minutes at 250 g in a pre-cooled centrifuge. Mix 20 microliters of trypan blue cell stain with cells in set one and count using a cell counter or hemocytometer. After removing the 15-milliliter tubes from the centrifuge, aspirate media from cell pellets and resuspend pellets in serum-free assay media to create a single-cell suspension.To reflate cells in 384-well and six-well plates, invert the 15-milliliter tubes several times for a homogenous cell suspension and transfer 500 microliters to 1.7 milliliter tubes in set two and set three. Add 0.5 microliters of DMSO to set two and 0.5 microliters of Halo 618 ligand to tube set three and mix well. Transfer the DMSO and ligand-positive cell suspensions to separate wells of reagent reservoirs.Use a multi-channel pipette to transfer 40 microliters of each suspension to the 384-well culture plate. Transfer the remaining cells to fresh six-well culture plates for subsequent western blot analysis and incubate both 384-well and six-well plates overnight at 37 degrees Celsius and 5%carbon dioxide. Take serum-free assay media prewarmed at 37 degrees Celsius.Dilute the thawed nanoluciferase substrate 1:100 in serum-free assay media and transfer it to a reagent reservoir. Using a multi-channel pipette, transfer 10 microliters of substrate mixture to each well of the 384-well culture plate. Gently rotate the plate for one minute.Insert the 384-well plate into a multi-mode plate reader and record the 460 and 618 nanometers emissions from all wells with cells. Calculate the raw BRET ratios for vehicle-positive and ligand-positive in milliBRET units using the given equation. Calculate the corrected BRET ratio by subtracting the vehicle-positive BRET ratio from that of the ligand-positive.The nano-CRAFS259A mutant reduces the BRET signal by approximately 45%and the nano-CRAFS621A mutant by around 25%The double mutant nearly eliminates the BRET signal.