FIGURE 5.

TRIM33 dynamically interacts with ALC1 in a PARP-dependent manner. A, HeLa cells were untreated (Un) or treated with IR or UV light, with or without PARP inhibitor (PARPi), and DNase-treated nuclear extracts were prepared at 5 and 10 min. TRIM33 IP was performed, followed by Western blotting (WB) using the indicated antibodies. IP, immunoprecipitation; No Ab, no antibody. B, quantitation of ALC1 interaction with TRIM33. The plot shows the ratio of the signal of ALC1 coimmunoprecipitation to ALC1 input. C, The FLAG WtALC1, ALC1K77R, and ALC1D723A mutants were expressed in 293 cells and subjected to UV irradiation. Protein extracts were prepared after 5 min and immunoprecipitated with anti-FLAG antibodies, followed by Western blotting using antibody against TRIM33 and FLAG. D, PAR-bound ALC1 does not bind TRIM33 in vitro. WtALC1, KR (ATPase dead) and DA (PAR binding mutant) ALC1 mutants and TRIM33 proteins were dot-blotted onto a nitrocellulose membrane and incubated with PAR, washed, and then incubated with purified TRIM33. Membranes were then processed for Western blot analysis with antibodies to PAR (top panel) or antibody to TRIM33 (bottom panel).