Fig. 8. hPARP-3 interacts with hPARP-1 at the centrosome. (A) Extracts from HeLa cells expressing either GST (lane a), GST-hPARP-3 (lanes b and c) or GST-N-ter hPARP-3 (lane d) were submitted to GST pull-down experiments, and the interacting proteins were analyzed by western blotting. When indicated, a treatment with 2 mM 3AB was applied for 2 hours before harvesting cell extracts. The blot was firstly probed with the mouse anti-hPARP-1 antibody (EGT69) (asterisks in upper panel) and subsequently with a polyclonal anti-GST antibody to reveal the proper expression of the fusion proteins (lower panel). (B) Sample of purified centrosomes (10⁷) separated on SDS-PAGE and analyzed by western blotting using successively antibodies against hPARP-1, hPARP-3 and -tubulin (lane e); purified recombinant hPARP-3 (50 ng) (lane f). (C) Subcellular localization of hPARP-1 in GFP-centrin expressing HeLa HC1 cells. hPARP-1 was detected using a monoclonal antibody (F1-23) followed by the anti-mouse fluor Alexa 568 conjugate (red). (D) Both hPARP-1 and hPARP-3, immunostained with their respective specific antibodies, are detected at the centrosome in HeLa cells. For all pictures, the magnifications are details of the area surrounding the arrowheads. Bars, 10 µm.