Ab74248

Liver samples were homogenized using Bio-Gen PRO200 Homogenizer (PRO Scientific) in RIPA buffer supplemented with 1 × Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), quantitated using a BCA protein assay (Pierce), and were separated on a 4–12% gradient SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane using iBlot Gel Transfer Device (Thermo Fisher Scientific). The membranes were blocked at room temperature for 30 min with blocking buffer containing 5% (w/v) nonfat dry milk in 1 × PBS and incubated with primary antibodies in blocking buffer at room temperature for 2 h or 4°C overnight. After washing three times with washing buffer (0.1% Tween-20 in 1 × PBS) for 5 min each wash, membranes were incubated with secondary antibodies in blocking buffer at room temperature for 1 h. After washing three times with washing buffer for 5 min each wash, proteins were detected based on Enhanced chemiluminescence (Abcam). Antibodies to cleaved PARP (ab32064), P21 (ab7960), HSP90 (ab74248), P54nrb (ab133574) and BIP (ab21685) were purchased from Abcam. Anti-PSF antibody was purchased from Sigma (p2860).