Immunoprecipitation and Western Blot Analysis

For immunoprecipitation and Western blotting [50 (link)], mouse anti-PARP-1 polyclonal antibody, mouse pan-Acetylation monoclonal antibody and mouse β-Actin monoclonal antibody was purchased from Proteintech (Proteintech Group, Chi cago, IL, USA). Mouse anti-PAR-monoclonal antibody was purchased from Trevigen (Trevigen Inc., Gaithersburg, Maryland, USA). Rabbit anti-sirtuin 3 (SIRT3) polyclonal antibody were purchased from Cell Signaling Technology (Beverly, MA, USA) and Proteintech (Proteintech Group, Chi cago, IL, USA). Nuclear proteins were extracted with a commercially available Nuclear and Cytoplasm Extraction kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s recommendations. Western blot analyses were performed as previously described [19 (link), 51 (link)] and β-Actin was used as a loading control. For co-IP, total proteins (400μg) incubated with 1μg anti-PARP-1 antibody for overnight (mouse normal IgG was used as a control), or nuclear proteins (200-300μg) incubated with 1 μg anti-SIRT3 antibody for overnight (rabbit normal IgG was used as a control), followed by 4h incubation with protein A/G PLUS-Agarose (Santa Cruz, CA, USA) at 4°C. The co-IP proteins were detected by Western blot.