Parp1 antibody

p65/RelA antibody (sc-372), α-tubulin antibody (H-300, sc-5546), and IgG (sc-2027) was from Santa Cruz Biotechnology, PARP1 antibody, histone H3 antibody (D1H2, #4499), and Cell Fractionation Kit (#9038) from Cell Signaling Technology, growth media, FBS, antibiotics, and Lipofectamine RNAiMAX from Life Technologies, IL-1β, IL6, TNFα and Z-VAD-fmk from Sigma Aldrich, H3K4me3 and acetyl-H4 antibody from Millipore, H3 antibody from Abcam, RANKL from Peprotech, siRNA from Qiagen. Corning Osteo Assay plates were from Corning. IL-1β neutralizing antibody was from R&D.

Proteins were extracted from test and control cells using Laemmli buffer containing a cocktail of protease inhibitors (Fermentas, cat. number R1329) and were quantified using a BCA protein assay kit (Pierce, USA, cat. number 23225). Protein extracts were separated on 12% SDS-PAGE gels and transferred onto a PVDF membrane. The membrane was blocked with 5% nonfat dry milk and incubated with a PARP-1 antibody (Cell Signaling Technology, USA) diluted in 3% nonfat dry milk overnight at 4°C [29 (link)]. The chosen antibody detects full-length and cleaved PARP-1 proteins (113 and 89 KDa, resp.) characteristic of apoptosis at those specific cleavage sites. The secondary antibody (i.e., alkaline phosphatase anti-Rabbit antibody) (KPL, USA) was diluted in 3% nonfat dry milk and incubated at room temperature for 30 minutes. PhosphaGlo substrate was used to detect chemiluminescent signals (KPL, USA). The membranes were stripped using a buffer containing β-mercaptoethanol and incubated for 15 min at 65°C. After stripping the membrane, the α-tubulin (loading control) (Pierce, USA) primary antibody was added (55 KDa) and the immunoblot was repeated.