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.

<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

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, ...

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Biology

7.2K Views.  Eye and ENT Hospital of Fudan University. Kleine ubiquitin-gerelateerde modifier (SUMO) familie eiwitten zijn geconjugeerd lysine residuen van doel eiwitten voor het regelen van diverse cellulaire processen. Dit witboek beschrijft een protocol voor de detectie van Retinoblastoom (Rb) eiwit SUMOylation onder endogene en exogene voorwaarden in menselijke cellen.

Video: 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.

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

Biochemistry

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.

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).

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.

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.

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

Measuring Ubiquitylated, SUMOylated, and ADP-Ribosylated DNA-Protein Crosslinks

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.

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

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-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.

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.

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

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine members that can monomethylate or symmetrically/asymmetrically dimethylate arginine residues. Several antibodies recognizing different types of arginine methylation of various proteins are available; thus, providing tools for the development of PRMT activity biomarker assays. PRMT antibody-based assays are challenging due to overlapping substrates and motif-based antibody specificities. These issues and the experimental setup to investigate the arginine methylation contributed by individual PRMTs are discussed. Through the careful selection of the representative substrates that are biomarkers for eight out of nine PRMTs, a panel of PRMT activity assays were designed. Here, the protocols for cellular assays quantitatively measuring the enzymatic activity of individual members of the PRMT family in cells are reported. The advantage of the described methods is their straightforward performance in any lab with cell culture and fluorescent western blot capabilities. The substrate specificity and chosen antibody reliability were fully validated with knockdown and overexpression approaches. In addition to detailed guidelines of the assay biomarkers and antibodies, information on the use of an inhibitor tool compound collection for PRMTs is also provided.

These protocols provide the methodology used to assess the enzymatic activity of individual members of the protein arginine methyltransferase (PRMT) family in cells. Detailed guidelines on assessing PRMT activity using endogenous and exogenous biomarkers, methyl-arginine recognizing antibodies, and inhibitor tool compounds are described.

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine ...

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

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