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.
