Name: in vivo detection and analysis of rb protein sumoylation in human cells
Uploaded: 2017-11-02T13:00:00
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?...

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+within+the+pRb+pathway:+is+there+a+code+of+conduct.">Diversity within the pRb pathway: is there a code of conduct.</a> Oncogene. 31 (40), 4343-4352 (2012).</li><li>Macdonald, J. I., Dick, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttranslational+modifications+of+the+retinoblastoma+tumor+suppressor+protein+as+determinants+of+function.">Posttranslational modifications of the retinoblastoma tumor suppressor protein as determinants of function.</a> Genes Cancer. 3 (11-12), 619-633 (2012).</li><li>Ezhevsky, S. 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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。

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		<col style="width:206pt" width="275" />
		<col style="width:89pt" width="119" />
		<col style="width:125pt" width="167" />
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	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">11995065</td>
			<td class="xl68" style="width:125pt" width="167"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">Opti-MEM&#160;</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">31985070</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Fetal Bovine Serum (FBS)</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">26140079</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Penicillin-Streptomycin</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">15140122</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Phosphate-buffered Saline (PBS)</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">10010023</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Trypsin-EDTA</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">25200056</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Thymidine</td>
			<td class="xl65" style="width:206pt" width="275">Sigma</td>
			<td class="xl70" style="width:89pt" width="119">T9250</td>
			<td class="xl68"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Nocodazole</td>
			<td class="xl65" style="width:206pt" width="275">Sigma</td>
			<td class="xl70" style="width:89pt" width="119">M1404</td>
			<td class="xl68"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">propidium iodide</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">P3566</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Triton X-100&#160;</td>
			<td class="xl65" style="width:206pt" width="275">AMRESCO</td>
			<td class="xl70" style="width:89pt" width="119">694</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">RNase A&#160;</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">EN0531</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">N-Ethylmaleimide</td>
			<td class="xl65" style="width:206pt" width="275">Sigma</td>
			<td class="xl70" style="width:89pt" width="119">E3876&#160;</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Sodium Dodecyl Sulfate (SDS)</td>
			<td class="xl65" style="width:206pt" width="275">AMRESCO</td>
			<td class="xl70" style="width:89pt" width="119">M107</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Nonidet P-40 Substitute (NP-40)</td>
			<td class="xl65" style="width:206pt" width="275">AMRESCO</td>
			<td class="xl70" style="width:89pt" width="119">M158</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">protease inhibitor</td>
			<td class="xl65" style="width:206pt" width="275">Roche</td>
			<td class="xl71" style="width:89pt" width="119">5892970001</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Mouse Immunoglobulin G (IgG)</td>
			<td class="xl65" style="width:206pt" width="275">Santa Cruz Biotechnology</td>
			<td class="xl71" style="width:89pt" width="119">sc-2025</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">Rb antibody</td>
			<td class="xl65" style="width:206pt" width="275">Cell Signaling Technology</td>
			<td class="xl71" style="width:89pt" width="119">#9309</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">Protein A/G-Sepharose Beads</td>
			<td class="xl65" style="width:206pt" width="275">Santa Cruz Biotechnology</td>
			<td class="xl71" style="width:89pt" width="119">sc-2003</td>
			<td class="xl68"></td>
		</tr>
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			<td class="xl68" height="21" style="height:15.75pt">Lipofectamine-2000&#160;</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl71" style="width:89pt" width="119">11668019</td>
			<td class="xl68"></td>
		</tr>
		<tr height="23" style="height:17.25pt">
			<td class="xl65" height="23" style="width:275pt;height:17.25pt" width="366">Nickel Nitrilotriacetic Acid (Ni-NTA) Agarose Beads</td>
			<td class="xl65" style="width:206pt" width="275">Qiagen</td>
			<td class="xl71" style="width:89pt" width="119">30230</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Imidazole</td>
			<td class="xl65" style="width:206pt" width="275">Sigma</td>
			<td class="xl71" style="width:89pt" width="119">I0250</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">4%-20% Gradient SDS-PAGE Gel</td>
			<td class="xl65" style="width:206pt" width="275">BIO-RAD</td>
			<td class="xl71" style="width:89pt" width="119">4561096</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">Polyvinylidene Difluoride (PVDF) Membrane</td>
			<td class="xl65" style="width:206pt" width="275">Millipore</td>
			<td class="xl71" style="width:89pt" width="119">IPVH00010</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Tween-20</td>
			<td class="xl65" style="width:206pt" width="275">AMRESCO</td>
			<td class="xl71" style="width:89pt" width="119">M147</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Tubulin antibody</td>
			<td class="xl65" style="width:206pt" width="275">Abmart</td>
			<td class="xl71" style="width:89pt" width="119">M30109</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">SUMO1 antibody</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl71" style="width:89pt" width="119">33-2044</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">GFP antibody</td>
			<td class="xl65" style="width:206pt" width="275">Abmart</td>
			<td class="xl71" style="width:89pt" width="119">M20004</td>
			<td class="xl68"></td>
		</tr>
		<tr height="23" style="height:17.25pt">
			<td class="xl65" height="23" style="width:275pt;height:17.25pt" width="366">Horseradish Peroxidase (HRP) secondary antibody</td>
			<td class="xl65" style="width:206pt" width="275">Jackson ImmunoResearch Laboratories</td>
			<td class="xl71" style="width:89pt" width="119">715-035-150</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">enhanced chemiluminescence (ECL) Kit</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl71" style="width:89pt" width="119">32106</td>
			<td class="xl68"></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, ...

Research

JoVE Journal

Biology

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

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

在体外和体内生物发光记者人类胚胎干细胞的基因显像

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

科研

生物学

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

微图案的表面上的蛋白质-蛋白质相互作用的体内检测

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Imaging Protein-protein Interactions in vivo

Imaging Protein-protein Interactions in vivo

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

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

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

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...