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

This study was supported by grants from the Science and Technology Commission of Shanghai (Grant No. 14411961800) and National Natural Science Foundation of China (Grant No. 81300805).

1. Detection of Endogenous Rb SUMOylation at the Early G1 Phase
<ol>
	<li>Cell culture and cell cycle synchronization.
	<ol>
		<li>Maintain HEK293 cells in growth medium containing Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 1% Pen-Strep and 10% fetal bovine serum (FBS) at 37 &#176;C and 5% CO2 in an incubator.</li>
		<li>Synchronize the HEK293 cells at the G0 phase.
		<ol>
			<li>Count the HEK293 cells using a hemocytometer and seed ~1.5 x&#160;107 cells in a 15...

<ol>
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This paper describes a protocol to detect and analyze the endogenous SUMOylation of Rb in human cells. As this method is specifically focused on the endogenous Rb protein without any alternation of global SUMO-related signal, it is an important tool for investigating Rb-SUMO modification under completely natural physiologic circumstances.
To achieve this aim, it is important to keep in mind that: 1) although SUMO comprises four isoforms (SUMO1-4, each encoded by different genes)in comparison t...

The accurate control of cell cycle progression in eukaryotic cells is based on a tight regulatory network, which ensures that particular events take place in an ordered manner1,2. One of the key players in this network is the retinoblastoma (Rb) protein, the first cloned tumor suppressor1,3. The Rb protein is thought to be a negative regulator of cell cycle progression, especially for the G0/G1 to S phase transition, and tumor growth4,5. Failure of Rb function either directly leads to the most common intraocular malignancy in children, retinoblastoma, or contributes to the development of many other types of cancer5. Moreover, Rb is involved in many cellular pathways including cell differentiation, chromatin remodeling, and mitochondria-mediated apoptosis3,6,7.
Post-translational modifications play a pivotal role in the regulation of RB function8,9. Phosphorylation is one such modification, and it usually leads to Rb inactivation. In quiescent G0 cells, Rb is active with a low phosphorylation level. As cells progress through G0/G1 phase, Rb is sequentially hyper-phosphorylated by a series of cyclin-dependent protein kinases (CDKs) and cyclins, such as cyclin E/CDK2 and cyclin D/CDK4/6, which inactivate Rb and eliminate its ability to repress cell-cycle related gene expression4,10. Rb could also be modified by small ubiquitin-related modifier (SUMO)11,12,13.
SUMO is a ubiquitin-like protein that is covalently attached to a variety of target proteins. It is crucial for diverse cellular processes, including cell cycle regulation, transcription, protein cellular localization and degradation, transport, and DNA repair14,15,16,17,18. The SUMO conjugation pathway consists of the dimeric SUMO E1 activating enzyme SAE1/UBA2, the single E2 conjugating enzyme Ubc9, multiple E3 ligases, and SUMO-specific proteases. Generally, nascent SUMO proteins must be proteolytically processed to generate the mature form. The mature SUMO is activated by the E1 heterodimer and then transferred to the E2 enzyme Ubc9. Finally, the C-terminal glycine of SUMO is covalently conjugated to the target lysine of a substrate, and this process is usually facilitated by E3 ligases. The SUMO protein can be removed from the modified substrate by specific proteases. A previous study by the authors of this paper revealed that SUMOylation of Rb increases its binding to CDK2, leading to hyper-phosphorylation at the early G1 phase, a process which is necessary for cell cycle progression13. We also demonstrated that the loss of Rb SUMOylation causes a decreased cell proliferation. Moreover, it was recently demonstrated that the SUMOylation of Rb protects the Rb protein from proteasomal turnover, thus increasing the level of Rb protein in cells19. Therefore, SUMOylation plays an important role in Rb function in various cellular processes. To further study the functional consequence and physiological relevance of Rb SUMOylation, it is important to develop an effective method to analyze the SUMO status of Rb in human cells or patient tissues.
SUMOylation is a reversible, highly dynamic process. Thus, it is usually difficult to detect the SUMO-modified proteins under completely endogenous conditions. This paper presents a method to detect endogenous Rb SUMOylation. Furthermore, it shows how to detect exogenous Rb SUMOylation of both wild-type Rb and its SUMO-deficient mutation11. In particular, Jacobs et al. described a method to increase the SUMO modification of a given substrate specifically by Ubc9 fusion-directed SUMOylation (UFDS)20. Based on this method, this protocol describes how to analyze the forced SUMOylation of Rb and its functional consequences. Given that hundreds of SUMO substrates have been described previously and more putative SUMO substrates have been identified from many proteomic-based assays, this protocol can be applied to analyze the SUMO-modification of these proteins in human cells.

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		<col style="width:275pt" width="366" />
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		<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>
		</tr>
		<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>
		</tr>
		<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>
		<tr height="21" style="height:15.75pt">
			<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

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

To detect endogenous Rb SUMOylation during cell cycle progression, this study first synchronized HEK293 cells at five different stages of the cell cycle (G0, early G1, G1, S, and G2/M) as described in the protocol section of this paper. The quality of synchronization was confirmed by using the nucleic acid stain with propidium iodide followed by flow cytometry analysis (Figure 1). Next, the cells were collected and lysed by denaturing RIPA buffer.The SUMO pro...

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

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

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

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

The overall goal of this experiment is to detect retinoblastoma proteins SUMOylation under endogenous and exogenous conditions in human cells. Small ubiquitin-related modifier, or SUMO, family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. Usually, it's more challenging to start a protein SUMOylation in cells due to the complexity of SUMOylation in vivo.This experiment detects the SUMOylation of both endogenous and exogenous Rb proteins. Though this method can provide insight into the SUMOylation of Rb proteins, it's also appropriate for the detection of many other SUMO target protein in cells. To begin, add 25 milliliters of DMEM containing one percent penicillin streptomycin, and incubate the HEK293 cells at 37 degrees Celsius for 72 hours.Wash the cells off the plate using three milliliters of ice cold PBS. Then transfer the cells into a new five milliliter tube. To obtain G1-phase cells at the end of the G0 synchronization process, remove the DMEM and add back fresh growth medium, thus allowing the HEK293 cells to re-enter the cell cycle.Then collect the cells at different G1 phases for cell cycle analysis and further SUMO assay. To synchronize the HEK293 cells at the S-phase, use the double thymidine block method. First, grow the HEK293 cells to a confluency of 50%Then wash the cells with pre-warmed PBS.Add growth medium supplemented with 2.5 millimolar thymidine for 18 hours as the first block. Remove the thymidine-containing medium and wash the cells twice with pre-warmed PBS. Add fresh growth medium for 14 hours to release the cells from the block.After releasing the cells from the first block, discard the medium with a pipette. Then add growth medium supplemented with 2.5 millimolar thymidine for another 18 hours as the second block before conducting an analysis of the cell cycle and Rb SUMOylation. For the G2, or M phase synchronization, plate approximately 15 million HEK293 cells on a 15-centimeter dish with 25 milliliters of growth medium, and grow for 24 hours.Then add nicotisol to the medium until a final concentration of 400 nanograms per milliliter is obtained. Finally, incubate the cells for 16 hours before conducting the analysis of the cell cycle by flow cytometry as described in the text protocol. Lyse the synchronized HEK293 cells by gently re-suspending them in one milliliter of ice cold radioimmunoprecipitation assay, or RIPA, lysis buffer containing freshly added isopeptidase inhibitor to block SUMO proteases and stabilize SUMO conjugates.Further homogenize the cells on ice by passing them through a 21 gauge needle attached to a two milliliter syringe 10 times. Then incubate the cells on ice for five minutes. Next, centrifuge the cell lysates at 18000 times G for 30 minutes at four degrees Celsius.Following centrifugation, transfer the supernatants to new 1.5 milliliter microcentrifuge tubes. To the supernatants, add one microgram of non-specific mouse immunoglobulin G of the same species and isotype as the monoclonal Rb antibody. Also, add 20 microliters of 50 percent protein AG sephyroslurry, then incubate the mixture for one hour at four degrees celsius with gentle rotation.Centrifuge the samples at three thousand times g for three minutes at four degrees celsius. Carefully collect the supernatants without disturbing the beads and transfer them to new one point five milliliter micro centrifuge tubes. To each of the samples, add five microliters of rb primary antibody and 40 microliters of 50 percent protein ag sephyroslurry then incubate the samples overnight at four degrees celsius with gentle rotation.After centrifuging, wash the beads with one milliliter of the rifa buffer. Mix the tubes well by rotation at four degrees celsius for 15 minutes. Then collect the beads by low speed centrifugation as before and discard the supernatants.After performing the wash four times, resuspend the beads in 30 microliters of one x sodium dodecalsulfate polyachrylymide gel electrophoresis loading buffer and mix well. Incubate the tubes at 100 degrees celsius in a heat block for 10 minutes. Centrifuge the samples at 12 thousand times g for one minute to pellet the beads.Finally, carefully collect the supernatants without disturbing the beads and transfer them to one point five milliliter microcentrifuge tubes. Perform cell culture transfection and cell lysis of HEK293 cells as described in the text protocol. Then wash 25 microliters of 50 percent nickel nicrolotriacetic acid agoros beads with ripa buffer in a one point five milliliter micro centrifuge tube for each sample.Collect the beads by centrifugation at three thousand times g for three minutes at four degrees celsius. Add one molar emytozol to each sample to obtain a final concentration of 10 millimolar. Then add each sample to the tube containing the prepared agoros beads.Incubate the beads and the lysates for two hours at four degrees celsius with gentle rotation. Then spin the beads at three thousand times g for three minutes at four degrees celsius. Following centrifugation, carefully remove the supernatants by pipeting.To avoid bead loss, do not aspirate the beads dry. Next, wash the beads with one milliliter of wash buffer containing 20 millimolar emytozol. Mix the tubes well by rotation at four degrees celsius for 15 minutes before collecting the beads by spinning.After the final wash, add 30 microliters of the Eleusian buffer containing 250 millimolar emitozol to each sample and flick to mix. Then incubate the samples for 20 minutes at four degrees celsius to elute the proteins. Mix each sample with six x SDS page loading buffer.Then incubate the tubes at 100 degrees celsius in a heat block for 10 minutes. Following centrifugation, load the immunoprecipitation or pull-down samples onto four percent to 20 percent gradient SDS page gels. Conduct electrophoresis at 120 volts for 90 minutes to separate the proteins.After the cell cycle synchronization and immunoprecipitation of the endogenous rb species, the presence of the SUMO signal was specifically detected by an anti SUMO one antibody at the early G1 phase. Representative results show the forced SUMOylation of the rb protein by directly fusing the SUMO E2 enzyme UBC9 to it's C terminal. Note the slowest migrating SUMOylated rb protein.By using the SUMO deficient mutant of rb, K720R, the presence of rb SUMOylation in cells is further confirmed. So in this procedure, many other SUMO target proteins can be analyzed in cultured cells. In most cases only a very small portion of a substrate is SUMOylated, thus while attempting this procedure it's important to optimize the efficiency of immunoprecipitation.

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