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 cm dish with 25ml growth medium for 24 h before treatment. After reaching a confluence of 70% - 80%, aspirate the medium and wash the cells twice with 5 mL prewarmed phosphate-buffered saline (PBS).</li>
			<li>Add 25 mL DMEM containing 1% Penicillin-Streptomycin and incubate the cells at 37 &#176;C for 72 h. Wash the cells off the plate using 3 mL ice-cold PBS. Transfer the cells into a new 5 mL tube.</li>
			<li>Subject a small portion of cells to cell cycle analysis by flow cytometry (step 1.2); collecting the remaining majority of the cells by centrifugation at 200 x g for 5 min at room temperature, and then store them in an ultra-lower temperature freezer until analysis of Rb SUMOylation.</li>
		</ol>
		</li>
		<li>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 (early G1: 30 min; G1: 2h) for cell cycle analysis and further SUMO assay.</li>
		<li>Synchronize the HEK293 cells at the S phase using the double-thymidine block method.
		<ol>
			<li>Grow HEK293 cells to a confluency of 50% and wash cells with prewarmed PBS. Then add growth medium supplemented with 2.5 mM thymidine for 18 h (first block).</li>
			<li>Remove the thymidine-containing medium, and then wash the cells twice with prewarmed PBS. Add fresh growth medium for 14 h to release the cells.</li>
			<li>Discard the medium with a pipette and add growth medium supplemented with 2.5 mM thymidine for another 18 h (second block) before conducting an analysis of the cell cycle and Rb SUMOylation.</li>
		</ol>
		</li>
		<li>For the G2/M synchronization, plate ~1.5 &#215; 107 HEK293 cells to a 15 cm dish with 25 mL growth medium for 24 h. Then add nocodazole to the medium until a final concentration of 400 ng/mL is obtained. Finally, incubate the cells for 16 h before conducting the analysis of the cell cycle and Rb SUMOylation.</li>
	</ol>
	</li>
	<li>Cell cycle analysis by flow cytometry.
	<ol>
		<li>Re-suspend the synchronized HEK293 in PBS, and then fix the cell suspension using ice-cold 70% ethanol for 2 h at 4 &#176;C. Note that to minimize cell clustering, add the cell suspension dropwise to the ice-cold 100% ethanol to obtain a final concentration of 70% ethanol while gently vortexing.</li>
		<li>Centrifuge the cells for 5 min at 500 x g, then carefully discard the supernatant and wash the cells twice with PBS. Repeat the centrifuge step.</li>
		<li>Add 500 &#181;L PBS containing 50 &#181;g/mL nucleic acid stain propidium iodide, 0.1% Triton X-100 and 1 &#181;g/mL RNase A to the cells and mix well. Incubate the cells for 15 min at 37 &#176;C.</li>
		<li>Store the samples at 4 &#176;C until analysis by flow cytometry.</li>
	</ol>
	</li>
	<li>Immunoprecipitation of endogenous Rb protein.
	<ol>
		<li>Prepare the HEK293 cell lysates.
		<ol>
			<li>Lyse the synchronized HEK293 cells by gently re-suspending them in 1 mL of ice-cold radio-immunoprecipitation assay (RIPA) lysis buffer (Table 1) containing freshly added 20 mM N-Ethylmaleimide, an isopeptidase inhibitor that could block SUMO proteases and stabilize SUMO conjugates.</li>
			<li>Further homogenize the cells on ice by Dounce homogenization, sonication or simply passing through 10 times through a 21 G needle attached on a 2 mL syringe. Then, incubate the cells on ice for 5 min. 
			NOTE: The anionic detergent sodium dodecyl sulfate (SDS) in the RIPA buffer is crucial for the later determination of the Rb-SUMO, as it could eliminate the unspecific SUMO signal that is derived from the non-covalent interaction between the Rb protein and other non-Rb SUMO-species.</li>
		</ol>
		</li>
		<li>Centrifuge the cell lysates at 18,000 x g for 30 min at 4 &#176;C. Transfer the supernatants to new 1.5 mL micro-centrifuge tubes. 
		NOTE: The protocol can be paused here. The proteins can be stored at -80 &#176;C for at least 6 months.</li>
		<li>To prepare the input control of each sample for the later Western blot, save a small portion of the above-described supernatant to a new tube and store at -80 &#176;C.</li>
		<li>To the above supernatants, add 1 &#181;g of non-specific mouse immunoglobulin G (IgG) of the same species and isotype as the monoclonal Rb antibody, and 20 &#181;L of 50% protein A/G-sepharose slurry. Then, incubate for 1 h at 4 &#176;C with gentle rotation.</li>
		<li>Centrifuge the samples at 3,000 x g for 3 min at 4 &#176;C. Carefully collect the supernatants without disturbing the beads, and transfer them to new 1.5 mL micro-centrifuge tubes. To each of the samples, add 5 &#181;L Rb primary antibody and 40 &#181;L of 50% protein A/G-sepharose slurry. Then, incubate overnight at 4 &#176;C with gentle rotation.</li>
		<li>Collect the beads by centrifugation at 3,000 x g for 3 min at 4 &#176;C, and carefully remove the supernatant by pipetting. Note that in order to avoid bead loss, do not aspirate the beads dry.</li>
		<li>Wash the beads four times with 1 mL of the RIPA buffer. Each time, mix the tubes well by rotation them at 4 &#176;C for 15 min. Collect the beads by low speed centrifugation at 3,000 x g for 3 min at 4 &#176;C and then discard the supernatants.</li>
		<li>After removing supernatants from the final wash, re-suspend the beads in 30 &#181;L 1x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (Table 1) and mix well.
		<ol>
			<li>Incubate the tubes at 100 &#176;C in a heat block for 10 min. Centrifuge the samples at 12,000 x g for 1 min to pellet the beads. Carefully collect the supernatants without disturbing the beads, and transfer them to 1.5 mL micro-centrifuge tubes.</li>
		</ol>
		</li>
		<li>Analyze the samples by 4% - 20% gradient SDS-PAGE gel and Western blot or store them at -20 &#176;C for later use.</li>
	</ol>
	</li>
</ol>
2. Analysis of 6XHis-tagged Exogenous Rb SUMOylation in Human Cells
<ol>
	<li>Cell culture and transfection.
	<ol>
		<li>Seed ~6 &#215; 106 HEK293 cells in a 10 cm dish and incubate for 24 h in growth medium under normal cell culture conditions to acquire 75%-85% confluent cells. Before transfection, remove the growth medium and add 6 mL of prewarmed reduced serum medium (see Table of Materials) and then place the dishes back into the incubator.</li>
		<li>For the Rb constitutive SUMOylation assay, add 15 &#181;g each of the 6XHis-tagged Rb-Ubc9-WT, Rb-Ubc9-C93S, Ubc9-WT and Ubc9-C93S plasmids in 500 &#181;L reduced serum medium in 1.5 mL micro-centrifuge tubes, respectively.
		<ol>
			<li>To analyze the SUMO-conjugation of wild type Rb and its SUMOylation-defective mutant, mix 10 &#181;g of either 6XHis-tagged Rb-WT or Rb-K720R plasmid together with GFP-SUMO1 (10 &#181;g) in 500 &#181;L reduced serum medium in 1.5 mL micro-centrifuge tubes13.</li>
		</ol>
		</li>
		<li>Meanwhile, in a separate tube, mix 50 &#181;L transfection reagent (see Table of Materials) in 500 &#181;L reduced serum medium, and incubate at room temperature for 5 min.</li>
		<li>Fully combine the two separate tubes described above, and incubate at room temperature for 15 min. Add the transfection reagent/plasmid complexes to the cells and continue to culture for 8 h at 5% CO2 and 37 &#176;C.</li>
		<li>Replace reduced serum medium with growth medium, and incubate the cells under normal culture conditions for 48 h after transfection.</li>
	</ol>
	</li>
	<li>Nickel affinity pull down of the 6XHis-tagged Rb protein.
	<ol>
		<li>Lyse the transfected HEK293 cells and extract the total proteins as described Section 1.3.1.</li>
		<li>Centrifuge the cell lysates at 18,000 x g for 30 min at 4 &#176;C. Carefully collect the supernatants and transfer them to clean tubes. 
		NOTE: The protein can be frozen at this point for more than 6 months at -80 &#176;C.</li>
		<li>To prepare the input control of each sample for the later Western blot, save a small portion of the supernatants described above to a new tube and store at -80 &#176;C.</li>
		<li>Wash 25 &#181;L of 50% nickel nitrilotriacetic acid (Ni-NTA) agarose beads with RIPA buffer twice in a 1.5 mL micro-centrifuge tube for each sample. Collect the beads by centrifugation at 3,000 x g for 3 min at 4 &#176;C.</li>
		<li>Add 1 M imidazole to each sample to obtain a final concentration of 10 mM. Then, add each sample to the tube containing the prepared Ni-NTA agarose beads.</li>
		<li>Incubate the beads and the lysates for 2 h at 4 &#176;C with gentle rotation. Spin the beads at 3,000 x g for 3 min at 4 &#176;C, and carefully remove the supernatants by pipetting. To avoid bead loss, do not aspirate the beads dry.</li>
		<li>Wash the beads with 1 mL of wash buffer containing 20 mM imidazole (Table 1). Each time, mix the tubes well by rotation at 4 &#176;C for 15 min. Collect the beads by spinning at 3,000 x g for 3 min at 4 &#176;C, and discard the supernatants.</li>
		<li>After the final wash, add 30 &#181;L of elution buffer containing 250 mM imidazole (Table 1) to each sample and flick to mix. Then, incubate for 20 min at 4 &#176;C to elute the proteins.</li>
		<li>Mix each sample with 6&#215; SDS-PAGE loading buffer (Table 1). Then, incubate the tubes at 100 &#176;C in a heat block for 10 min.</li>
		<li>Centrifuge at 12,000 x g for 1 min to pellet the beads. Carefully collect the supernatants without disturbing the beads, and transfer the samples to 1.5 ml micro-centrifuge tubes. The samples can be analyzed by Western blot or stored at -20 &#176;C for later use.</li>
	</ol>
	</li>
</ol>
3. Western Blot
<ol>
	<li>Load the immunoprecipitation or pull down samples obtained from the previous steps onto 4-20% gradient SDS-PAGE gels. Conduct electrophoresis at 120 V for 90 min to separate the proteins.</li>
	<li>Transfer the proteins from the gel to a polyvinylidene difluoride (PVDF) membrane by electroblotting at 300 mA for 90 min at 4 &#176;C using the tank transfer method. Block the PVDF membrane in block buffer containing 5% nonfat milk (Table 1) for 1 h at room temperature.</li>
	<li>Incubate the membrane with primary antibody in antibody dilution buffer containing 3% bovine serum albumin (BSA) (Table 1) overnight at 4 &#176;C. For primary antibodies, use a working concentration of 0.5 &#181;g/mL (anti-SUMO1 antibody), or a dilution of 1:2000 (anti-Rb and anti-Tubulin antibodies), or 1:5,000 (anti-GFP and anti-His antibody).</li>
	<li>Wash the membrane 3x for 10 min each time using 1x tris-buffered saline with Tween (TBST) buffer (Table 1). Incubate the membrane with species specific Horseradish Peroxidase (HRP)-conjugated secondary antibodies diluted 1:5,000 in block buffer containing 5% nonfat milk (Table 1). Wash the membrane 3x for 10 min each time using 1x TBST buffer.</li>
	<li>Incubate the membrane with enhanced chemiluminescence (ECL) working solution. Cover the membrane with plastic wrap and expose it to X-ray film depending on the strength of signal.</li>
</ol>

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The authors have nothing to disclose.

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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	<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>
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			<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>
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		<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...

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

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, ...

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

Research

JoVE Journal

Biology

7.2K Views.  Eye and ENT Hospital of Fudan University. 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. 

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

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

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 software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

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

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

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

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

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

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

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

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

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

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

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

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

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

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

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

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 include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

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

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

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

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.

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

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

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

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

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Rozdziały w tym wideo

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

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

Imaging Protein-protein Interactions in vivo

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

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

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

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

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

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

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