Name: in vivo detection and analysis of rb protein sumoylation in human cells
Uploaded: 2017-11-02T13:00:00.000+00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells at JoVE.com

这项研究得到了上海科学技术委员会 (grant No. 14411961800) 和中国国家自然科学基金 (grant No. 81300805) 的资助。

1. 在早期 G1 阶段检测内源性 Rb sumo <ol> <li> 单元格区域性和单元格周期同步. 
	<ol> <li> 在含有 Dulbecco 和 #39 的生长培养基中维护 HEK293 细胞 (DMEM), 辅以1% 笔链球菌和10% 胎牛血清 (FBS) 在37和 #176; C 和 5% CO 2 在孵化器中. </li>
		<li> 在 G0 阶段同步 HEK293 单元格。
		<ol> <li> 使用例和种子 ~ 1.5 x 和 #160 的 HEK293 单元格计数; 10 7 单元格在 15 cm 盘中, 在治疗前为24小时的25ml 生长培养基。在达到 70%-80% 的汇合后, 吸气培养基, 用5毫升 prewarmed 磷酸缓冲盐水 (PBS) 冲洗两次细胞. </li>
			<li> 添加25毫升 DMEM, 含有1% 青霉素-链霉素, 并孵育细胞在37和 #176; C 为 72 h. 用3毫升 ice-cold PBS 清洗板上的细胞。将细胞转化为新的5毫升管. </li>
			<li> 用流式细胞仪对细胞周期进行分析 (步骤 1.2); 在室温下以 200 x g 的离心法收集剩余的大多数细胞, 然后将它们储存在 tme 温度的冷冻库中, 直到分析Rb sumo. </li>
		</ol> </li> <li> 获取 G1-phase 单元格, 在 G0 同步过程结束时, 移除 DMEM 并添加回新的生长介质, 从而允许 HEK293 单元重新进入细胞周期。然后, 在不同的 G1 阶段收集细胞 (早 G1:30 分钟;G1: 2h) 用于细胞周期分析和进一步的相扑检测. </li>
		<li> 使用 double-thymidine 块方法在 S 阶段同步 HEK293 单元格。
		<ol> <li> 将 HEK293 单元格增长为 70-100 50%, 并使用 prewarmed PBS 清洗细胞。然后加入生长培养基补充2.5 毫米胸苷为 18 h (第一块). </li>
			<li> 取下含有胸苷的培养基, 然后用 prewarmed PBS 冲洗两次细胞。添加新鲜培养基 14 h 释放细胞. </li>
			<li> 在对细胞周期和 Rb sumo 进行分析之前, 用吸管将培养基与 2.5 mM 的胸苷添加到另一 18 h (第二块) 中. </li>
		</ol> </li> <li> 用于 G2/M 同步、板 ~ 1.5 和 #215; 10 7 HEK293 细胞到 15 cm 盘与25毫升成长媒介 24 h。然后在培养基中加入 nocodazole, 直到最终浓度达到 400 ng/毫升。最后, 在对细胞周期和 Rb sumo 进行分析之前, 先孵育16小时的细胞. </li>
	</ol> </li> <li> 流式细胞术的细胞周期分析. 
	<ol> <li> 在 PBS 中重新挂起同步 HEK293, 然后在4和 #176 中使用 ice-cold 70% 乙醇对2小时内的单元悬浮进行修复; c. 注意到为了最小化细胞聚类, 在 ice-cold 100% 乙醇中加入细胞悬浮滴以获得最终浓度70% 乙醇, 同时轻轻涡流. </li>
		<li> 将细胞离心5分钟, 500 x g, 然后小心地丢弃上清液, 用 PBS 冲洗两次细胞。重复离心机步骤. </li>
		<li> 添加500和 #181; 包含50和 #181 的 PBS; g/毫升核酸染色碘碘化物, 0.1% 海卫 X-100 和1和 #181; g/毫升核糖核酸 A 对细胞和混合好。在37和 #176 孵育细胞15分钟; C. </li>
		<li> 将样品存储在4和 #176; C 直到流式细胞仪进行分析. </li>
	</ol> </li> <li> 沉淀内生 Rb 蛋白。
	<ol> <li> 准备 HEK293 单元格裂解。
		<ol> <li> 通过在1毫升 ice-cold 无线电沉淀检测 (帕) 裂解缓冲区 ( 表 1 ) 中轻轻 re-suspending, 溶解同步 HEK293 细胞, 其中含有新添加的20毫米 n-Ethylmaleimide, 一种 isopeptidase 抑制剂, 可以阻止相扑蛋白酶和稳定的相扑共轭. </li>
			<li> 通过在2毫升注射器上附着的21克针, 进一步质冰上的细胞 Dounce 均匀化, 超声或简单地通过10次。然后, 在冰上孵育细胞5分钟. 
			注: 阴离子洗涤剂十二烷基硫酸钠 (SDS) 在帕缓冲是关键的后期确定的 rb-相扑, 因为它可以消除不相扑信号是衍生的价相互作用的 rb 蛋白和其他非 Rb 相扑种. </li>
		</ol> </li> <li> 在4和 #176 将单元格裂解为 1.8万 x g, 30 分钟; c. 将清转移到新的1.5 毫升微离心管. 
		注意: 协议可以在此处暂停。蛋白质可以储存在-80 和 #176; C 至少6月. </li>
		<li> 为以后的西部印迹准备每个样品的输入控制, 保存上面描述的上清的一小部分到新的管子和商店在-80 和 #176; C. </li>
		<li> 对上述清, 添加1和 #181; g 的非特异性小鼠免疫球蛋白 g (IgG) 的同一物种和同种的单克隆 Rb 抗体, 和20和 #181; L 50% 蛋白 A/g-琼脂浆。然后, 在4和 #176 孵育1小时; C 温和旋转. </li>
		<li> 离心样品在 3000 x g 为3分钟在4和 #176; c. 小心地收集清不干扰的珠子, 并转移到新的1.5 毫升微离心管。对每个样品, 增加5和 #181; i Rb 主要抗体和40和 #181; l 50% 蛋白 A/G-琼脂浆。然后, 在4和 #176 一夜之间孵育; C 温和旋转. </li>
		<li> 通过离心 3000 x g 收集珠子3分钟, 在4和 #176; C, 并小心地清除上清移。注意, 为了避免珠损, 不要将珠子吸干. </li>
		<li> 用1毫升的帕缓冲液洗涤珠子四次。每一次, 通过旋转他们在4和 #176 的管; c 为 15 min. 用低速离心法在 3000 x g 处收集珠子, 在4和 #176; c, 然后丢弃清. </li>
		<li> 从最后的洗涤中除去清后, 在30和 #181 中 re-suspend 珠; 1x 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (SDS 页) 加载缓冲区 ( 表 1 ) 并混合好。
		<ol> <li> 在100和 #176 中孵育管; C 在热块中进行10分钟离心, 将样品在 1.2万 x g 为1分钟, 将珠子颗粒。仔细收集清不干扰珠子, 并转移到1.5 毫升微离心管. </li>
		</ol> </li> <li> 通过 4%-20% 梯度 SDS-页凝胶和西式印迹分析样品, 或将它们存储在-20 和 #176; C 用于以后使用. </li>
	</ol> </li> </ol> 2。6 xhis 标记外源 Rb sumo 在人类细胞中的分析 <ol> <li> 单元格区域性和转染. 
	<ol> <li> 种子 ~ 6 和 #215; 10 6 HEK293 在 10 cm 盘中的细胞, 在正常细胞培养条件下孵育 24 h, 以获得 75%-85% 的汇合细胞。在转染前, 移除培养基, 加入6毫升的 prewarmed 降低血清培养基 (见 材料表 ), 然后将这些盘子放回孵化器. </li>
		<li> 对于 Rb 本构 sumo 测定, 增加15和 #181; g 在500和 #181 中分别添加了 6 xhis 标记的 Rb-Ubc9-WT、Rb-Ubc9-C93S、Ubc9-WT 和 Ubc9-C93S 质粒; 在1.5 毫升微离心管中降低了血清培养基。
		<ol> <li> 分析野生型 Rb 及其 sumo 缺陷突变体的相扑共轭, 混合10和 #181; g 6 xhis 标记的 Rb 或 Rb-K720R 质粒连同 GFP-SUMO1 (10 和 #181; g) 在500和 #181; 在 1.5 mL 微离心机中降低血清培养基管 13 . </li>
		</ol> </li> <li> 同时, 在一个单独的管中, 混合50和 #181; l 转染试剂 (见 材料表 ) 在500和 #181; l 降低血清培养基, 室温孵育5分钟。
		<li> 完全结合上述两个单独的管, 并在室温下孵育15分钟. 将转染试剂/质粒配合物添加到细胞中, 并继续培养8h 在 5% CO 2 和37和 #176; C. </li>
		<li> 用培养基取代还原培养基, 在正常培养条件下孵育细胞48小时后转染. </li>
	</ol> </li> <li> 镍亲和力拉下 6 xhis 标记的 Rb 蛋白. 
	<ol> <li> 溶解转染的 HEK293 细胞并提取总蛋白, 如描述的 1.3.1. </li>
		<li> 离心机裂解在 1.8万 x g 的30分钟, 在4和 #176; c. 仔细收集清和转移到清洁管. 
		注: 在这一点上, 蛋白质可以冻结在6月以上的-80 和 #176; C. </li>
		<li> 为以后的西部印迹准备每个样本的输入控制, 将上面描述的清的一小部分保存到一个新的管道上, 并存储在-80 和 #176; C. </li>
		<li> 洗涤25和 #181; 50% 镍三酸 (Ni-NTA) 琼脂糖珠与帕缓冲两次在1.5 毫升微离心管为每个样品。收集的珠子离心 3000 x g 为3分钟, 在4和 #176; C. </li>
		<li> 将1米咪唑添加到每个样品中以获得最终浓度10毫米。然后, 将每个样品添加到含有制备的 NTA 琼脂糖珠的试管中. </li>
		<li> 在4和 #176 上孵育珠子和裂解的2小时; C 温和旋转。旋转珠子在 3000 x g 为3分钟在4和 #176; C, 并且小心地去除清由移。为避免珠损, 请勿将珠子吸干. </li>
		<li> 用1毫升洗涤缓冲液洗涤含有 20 mM 咪唑的珠子 ( 表 1 )。每一次, 在4和 #176 的旋转, 混合管; c 为15分钟, 在 3000 x g 处旋转3分钟, 在4和 #176; c, 并丢弃清. </li>
		<li> 最后一次洗涤后, 添加30和 #181; 洗脱缓冲液中含有250毫米咪唑 ( 表 1 ), 每个样品和轻弹混合。然后, 孵育20分钟, 在4和 #176; C 洗蛋白质. </li>
		<li> 将每个示例与6和 #215 混合; SDS-页加载缓冲区 ( 表 1 )。然后, 在100和 #176 的试管中孵育; C 在热块中为 10 min. </li>
		<li> 离心机在 1.2万 x g 为1分钟颗粒的珠子。仔细收集清不干扰珠子, 并转移样品到1.5 毫升微离心管。样品可以用印迹或-20 和 #176 的方法储存, 以备以后使用. </li>
	</ol> </li> </ol> 3。西部印迹 <ol> <li> 将从先前步骤中获得的沉淀或拉下的样本加载到4-20% 渐变 SDS 页凝胶中。在120伏进行电泳90分钟分离蛋白质. </li>
	<li> 将 electroblotting 在300毫安的氟 (PVDF) 膜上的蛋白质从凝胶中转移到90分钟, 在4和 #176; C 使用坦克转移法。在室温下, 在含有5% 脱脂牛奶 ( 表 1 ) 的块缓冲中, 将 PVDF 膜阻挡在1小时内. </li>
	<li> 在4和 #176 过夜的抗体稀释缓冲液中孵育含有3% 牛血清白蛋白 (BSA) ( 表 1 ) 的原抗体的膜;对于主要抗体, 使用0.5 和 #181 的工作浓度; g/毫升 (anti-SUMO1 抗体), 或稀释 1:2000 (抗 Rb 和抗蛋白抗体), 或 1:5, 000 (抗 GFP 和抗他的抗体). </li>
	<li> 每次使用1x 的三缓冲盐水 (TBST) 缓冲 ( 表 1 ) 将膜冲洗10分钟。用种特异性辣根过氧化物酶 (HRP) 孵育膜, 在含有5% 脱脂牛奶 ( 表 1 ) 的块缓冲液中稀释1:5、000的共轭二抗体。每次使用 1x TBST 缓冲液清洗膜 3x 10 分钟. </li>
	<li> 用增强化学发光 (ECL) 工作溶液孵育膜。用塑料包覆膜, 根据信号强度将其暴露在 x 光胶片上. </li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SUMO+pathway:+emerging+mechanisms+that+shape+specificity,+conjugation+and+recognition.">The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition.</a> Nat Rev Mol Cell Biol. 11 (12), 861-871 (2010).</li><li>Bernier-Villamor, V., Sampson, D. A., Matunis, M. J., Lima, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+E2-mediated+SUMO+conjugation+revealed+by+a+complex+between+ubiquitin-conjugating+enzyme+Ubc9+and+RanGAP1.">Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1.</a> Cell. 108 (3), 345-356 (2002).</li><li>Saitoh, H., Hinchey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+of+small+ubiquitin-related+protein+modifiers+SUMO-1+versus+SUMO-2/3.">Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3.</a> J Biol Chem. 275 (9), 6252-6258 (2000).</li><li>Ayaydin, F., Dasso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+in+vivo+dynamics+of+vertebrate+SUMO+paralogues.">Distinct in vivo dynamics of vertebrate SUMO paralogues.</a> Mol Biol Cell. 15 (12), 5208-5218 (2004).</li></ol>

作者没有什么可透露的。

本文介绍了一种检测和分析人体细胞 Rb 的内源性 sumo 的协议。由于这种方法是专门针对内源性 rb 蛋白没有任何变化的全球相扑相关的信号, 它是一个重要的工具, 调查 Rb-相扑在完全自然的生理环境。
为了达到这个目的, 重要的是要记住: 1) 虽然相扑包括四个异构体 (SUMO1-4, 每个编码不同的基因) 相比, 泛, 所有的相扑物种都少得多;2) 对于大多数相扑靶蛋白, 只有一小部分的特定?...

真核细胞周期进展的精确控制是基于严密的监管网络, 确保特定事件以有序的方式1,2进行。在这个网络的关键球员之一是视网膜母细胞瘤 (Rb) 蛋白, 第一个克隆肿瘤抑制1,3。Rb 蛋白被认为是细胞周期进展的负调节因子, 尤其是对 G0/G1 S 相变和肿瘤生长4,5。Rb 功能的失败直接导致儿童、视网膜母细胞瘤的最常见的眼内恶性肿瘤, 或有助于许多其他类型的癌症的发展5。此外, Rb 涉及许多细胞通路, 包括细胞分化, 染色质重塑, 和线粒体介导的凋亡3,6,7。
后平移修改在 RB 函数的调节中起着关键作用8,9。磷酸化是一个这样的修改, 它通常导致 Rb 失活。在静止 G0 细胞, Rb 是活跃的低磷酸化水平。随着细胞在 G0/G1 阶段的进展, rb 是连续的高磷酸化的一系列周期素依赖性蛋白激酶 (激酶) 和周期, 例如, 细胞周期素 E/CDK2 和 D/CDK4/6, 使 rb 失效, 并消除它的能力, 抑制电池周期相关基因表达式4,10。Rb 也可以通过小泛素相关修饰符 (相扑)11,12,13进行修改。
相扑是一种泛素状的蛋白质, 它与各种靶蛋白的共价键相连。它是关键的不同的细胞过程, 包括细胞周期调节, 转录, 蛋白质细胞定位和降解, 运输和 DNA 修复14,15,16,17,18. 相扑共轭通路包括二相扑 E1 活化酶 SAE1/UBA2, 单 E2 共轭酶 Ubc9, 多 E3 酶, 和相扑特有的蛋白酶。一般来说, 新生的相扑蛋白必须 proteolytically 加工, 以产生成熟的形式。成熟的相扑被激活的 E1 heterodimer, 然后转移到 E2 酶 Ubc9。最后, 相扑的 c-端甘氨酸与基材的靶赖氨酸共价共轭, 这一过程通常由 E3 酶。相扑蛋白可以通过特定的蛋白酶从修饰的基质中除去。本文作者先前的一项研究表明, Rb 的 sumo 增加了它与 CDK2 的结合, 导致早期 G1 阶段的超磷酸化, 这是细胞周期进程13所必需的过程。我们还证明, Rb sumo 的损失导致细胞增殖减少。此外, 最近证明, rb 的 sumo 保护 rb 蛋白从体的营业额, 从而提高了 rb 蛋白的水平, 在细胞19。因此, sumo 在各种细胞过程的 Rb 功能中起着重要的作用。为了进一步研究 rb sumo 的功能后果和生理相关性, 必须建立一种有效的方法来分析 rb 在人细胞或患者组织中的作用。
sumo 是一个可逆的、高度动态的过程。因此, 在完全内源性条件下, 通常很难检测到相扑修饰的蛋白质。本文提出了一种检测内源 Rb sumo 的方法。此外, 还说明了如何检测野生型 rb 的外源 rb sumo 和其相扑缺陷突变11。特别是, 雅各布斯et al.描述了一种方法来增加特定基板的相扑修饰, 具体由 Ubc9 融合定向 sumo (ufd)20。基于这种方法, 本协议描述了如何分析 Rb 的强迫 sumo 及其功能性后果。考虑到数百个相扑基质已经被描述过, 而且更多的假定相扑基质已经从许多 proteomic-based 的检测中发现, 这个协议可以用来分析这些蛋白质在人类细胞中的相扑修饰。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher Scientific</td><td>11995065</td><td></td></tr><tr><td>Opti-MEM&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>31985070</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Thermo Fisher Scientific</td><td>26140079</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Phosphate-buffered Saline (PBS)</td><td>Thermo Fisher Scientific</td><td>10010023</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Thermo Fisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Thymidine</td><td>Sigma</td><td>T9250</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma</td><td>M1404</td><td></td></tr><tr><td>propidium iodide</td><td>Thermo Fisher Scientific</td><td>P3566</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>AMRESCO</td><td>694</td><td></td></tr><tr><td>RNase A&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>EN0531</td><td></td></tr><tr><td>N-Ethylmaleimide</td><td>Sigma</td><td>E3876&amp;nbsp;</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>AMRESCO</td><td>M107</td><td></td></tr><tr><td>Nonidet P-40 Substitute (NP-40)</td><td>AMRESCO</td><td>M158</td><td></td></tr><tr><td>protease inhibitor</td><td>Roche</td><td>5892970001</td><td></td></tr><tr><td>Mouse Immunoglobulin G (IgG)</td><td>Santa Cruz Biotechnology</td><td>sc-2025</td><td></td></tr><tr><td>Rb antibody</td><td>Cell Signaling Technology</td><td>#9309</td><td></td></tr><tr><td>Protein A/G-Sepharose Beads</td><td>Santa Cruz Biotechnology</td><td>sc-2003</td><td></td></tr><tr><td>Lipofectamine-2000&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11668019</td><td></td></tr><tr><td>Nickel Nitrilotriacetic Acid (Ni-NTA) Agarose Beads</td><td>Qiagen</td><td>30230</td><td></td></tr><tr><td>Imidazole</td><td>Sigma</td><td>I0250</td><td></td></tr><tr><td>4%-20% Gradient SDS-PAGE Gel</td><td>BIO-RAD</td><td>4561096</td><td></td></tr><tr><td>Polyvinylidene Difluoride (PVDF) Membrane</td><td>Millipore</td><td>IPVH00010</td><td></td></tr><tr><td>Tween-20</td><td>AMRESCO</td><td>M147</td><td></td></tr><tr><td>Tubulin antibody</td><td>Abmart</td><td>M30109</td><td></td></tr><tr><td>SUMO1 antibody</td><td>Thermo Fisher Scientific</td><td>33-2044</td><td></td></tr><tr><td>GFP antibody</td><td>Abmart</td><td>M20004</td><td></td></tr><tr><td>Horseradish Peroxidase (HRP) secondary antibody</td><td>Jackson ImmunoResearch Laboratories</td><td>715-035-150</td><td></td></tr><tr><td>enhanced chemiluminescence (ECL) Kit</td><td>Thermo Fisher Scientific</td><td>32106</td><td></td></tr></tbody></table>

in vivo detection and analysis of rb protein sumoylation in human cells

蛋白质的修饰修饰对细胞内信号转导的适当调节至关重要。在这些修饰中, 小泛素相关的修饰剂 (相扑) 是一种泛素样蛋白, 它是以共价键的方式附着在各种靶蛋白的赖氨酸残留物上, 以调节细胞过程, 如基因转录、DNA 修复、蛋白质相互作用和退化, 亚细胞传输和信号转导。最常见的检测蛋白质 sumo 的方法是基于基因重组标记蛋白在细菌中的表达和纯化, 允许一个体外生化反应, 这是简单的, 适合解决机械问题.然而, 由于 sumo 的过程的复杂性在体内, 它是更具挑战性的检测和分析蛋白质 sumo 的细胞, 特别是在内源条件下。本文作者最近的一项研究表明, 内源性视网膜母细胞瘤 (Rb) 蛋白, 是一个肿瘤抑制物, 这是至关重要的消极调节的周期进展, 是专门 SUMOylated 在早期 G1 阶段。本文介绍了一种在细胞内源和外源条件下检测和分析 Rb sumo 的协议。本议定书适用于型和功能研究的相扑-修改的 Rb, 以及许多其他相扑靶蛋白, 在人类细胞。

为了在细胞周期进展过程中检测内源性 Rb sumo, 本研究首先在五不同的细胞周期 (G0、早期 G1、G1、S 和 G2/M) 中同步 HEK293 细胞, 如本文的协议部分所述。使用碘碘化物的核酸染色和流式细胞仪分析 (图 1) 确认了同步的质量。其次, 通过变性帕缓冲液收集和裂解细胞。相扑蛋白酶抑制剂, n-Ethylmaleimide, 被添加到最后浓度20毫米, 以保存本地相扑信号在实验。...

Watch this Scientific Journal Video about In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells at JoVE.com

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

小泛素相关的修饰剂 (相扑) 家族蛋白被共轭到靶蛋白的赖氨酸残留物来调节各种细胞过程。本文介绍了一种检测视网膜母细胞瘤 (Rb) 蛋白 sumo 的方法。

在体内人细胞 Rb 蛋白 sumo 的检测与分析

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

in-vivo-detection-analysis-rb-protein-sumoylation-human

Research

JoVE Journal

Biology

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

7.2K 次观看.  Eye and ENT Hospital of Fudan University. 小泛素相关的修饰剂 (相扑) 家族蛋白被共轭到靶蛋白的赖氨酸残留物来调节各种细胞过程。本文介绍了一种检测视网膜母细胞瘤 (Rb) 蛋白 sumo 的方法。

视频: In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

科研

生物化学

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.

荧光苏门答腊蛋白对 sumo 修饰蛋白的定位

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

SUMO 结合实体 （SUBEs） 作为肝癌中 SUMO 蛋白酶的浓缩、分离、识别和表征的工具

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

蛋白泛素化的检测

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

生物学

测量泛素化、SUMO 化和 ADP 核糖基化 DNA-蛋白质交联

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

作者聚焦: DNA 蛋白交联及其翻译后修饰的定量检测  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

在体内人细胞 Rb 蛋白 sumo 的检测与分析

摘要

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此视频中的章节

RNA 结合区质量谱鉴定的样品制备

荧光苏门答腊蛋白对 sumo 修饰蛋白的定位

SUMO 结合实体（SUBEs）作为肝癌中 SUMO 蛋白酶的浓缩、分离、识别和表征的工具

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

蛋白泛素化的检测

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

蛋白质纯化技术，可以检测SUMO化修饰和芽殖酵母蛋白动粒和Ndc10泛素Ndc80的

在体内人细胞 Rb 蛋白 sumo 的检测与分析

摘要

探索更多视频

此视频中的章节

RNA 结合区质量谱鉴定的样品制备

荧光苏门答腊蛋白对 sumo 修饰蛋白的定位

SUMO 结合实体 （SUBEs） 作为肝癌中 SUMO 蛋白酶的浓缩、分离、识别和表征的工具

分析泛性基质和泛性基质样的依从性转化后修饰和重大改变的识别

蛋白泛素化的检测

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

DNA-蛋白质交联及其翻译后修饰的定量检测

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

蛋白质纯化技术，可以检测SUMO化修饰和芽殖酵母蛋白动粒和Ndc10泛素Ndc80的

SUMO 结合实体（SUBEs）作为肝癌中 SUMO 蛋白酶的浓缩、分离、识别和表征的工具