Detecting Protein Modifications in HeLa Cells

HeLa cells at 70–80% confluency, transfected with the indicated plasmids or treated with H₂O₂, were washed with 1 × phosphate-buffered saline (PBS) and resuspended in RIPA buffer (1 × PBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) along with 1 × protease inhibitor cocktail and phosphatases inhibitor cocktail (Roche Applied Science). To detect PML SUMOylation in HeLa cells, whole-cell extracts in the presence of NEM were prepared. Lysed cells were centrifuged at 4°C at 12 000 r.p.m. for 10 min, and the supernatant was incubated with protein A-conjugated beads for preclearing. To detect protein–protein interactions or acetylation and SUMO1 modification of PML, whole-cell extracts were incubated with anti-HA antibody-conjugated beads (Sigma F2426) or anti-FLAG antibody-conjugated beads (Sigma E6779) for 2 h. The beads were washed with NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, and 0.1% NP-40) five times, and supernatants were discarded. Then, 2 × sample buffer was added to the beads, followed by SDS-PAGE and western blotting as previously described.^{9 (link), 10 (link)}