Fig. 5. B23 interacts constitutively with PARP-1 and PARP-2 through their DNA binding domains and PARP-1 BRCT domain. (A) Schematic representation of GST-hPARP-1 and GST-hPARP-2. DBD, DNA binding domain. GST (lane 7), GST-hPARP-1 (lanes 2-6) or GST-hPARP-2 (lanes 8-12) were overexpressed in HeLa cells either untreated (lanes 2, 5, 8 and 11) or treated with 1 mM H₂O₂ for 10 minutes (lanes 3 and 9), 4 mM 3-AB for 3 hours (lanes 4 and 10) or 0.2 µg/ml actinomycin D for 3 hours (ActD, lanes 6 and 12). 10 µg/ml ethidium bromide was added to the lysate (EtBr, lanes 5 and 11). Proteins were analyzed by GST pull down and western blot using anti-B23 and anti-GST antibodies as indicated in the lower panel. Lane 1: input corresponding to 1/30th of the lysate. (B) GST (lanes 2 and 9), GST mPARP-2 (lane 10) or GST-tagged deletion mutants of hPARP-1 (lanes 3-7) or of mPARP-2 (lanes 11-13) were expressed in HeLa cells. Interacting proteins were analyzed by GST pull down and western blotting with anti-B23 and anti-GST antibodies. Input (lanes 1 and 8): 1/30th of the GST-expressing cell lysate. (C) Immunoprecipitation with anti-GFP antibodies of GFP (lane 2), mPARP-2_1-69-GFP (lane 3) and mPARP-2_1-69A_4-7GFP (lane 4) overexpressed in HeLa cells. Western blots were subsequently probed with a monoclonal anti-B23 antibody (top) and a polyclonal anti GFP antibody. Lane 1: crude extract from 10⁵ HeLa cells.