Mouse monoclonal cytochrome c

For western blotting, tissue lysate was prepared using RIPA buffer (Cell signaling Technology, USA) with protease inhibitors cocktail (Roche Diagnostics, Mannheim, Germany). The lysates were centrifuged at 14,000 × g for 20 min at 4°C. The protein content of the supernatant was determined with the Bradford protein assay (Sigma, USA). Forty micrograms protein was resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) after denaturing in sample buffer and transferred onto polyvinylidene fluoride membranes (PVDF; Thermo Fisher Scientific, USA). After blocking with 5% BSA, the blots were probed with the following antibodies: mouse monoclonal α-SMA (1 : 300; Santa Cruz Biotechnology), mouse monoclonal beta actin (1 : 1000; Santa Cruz Biotechnology), mouse monoclonal collagen 1A1 (1 : 300; Santa Cruz Biotechnology), mouse monoclonal anti Bax (1 : 300; Santa Cruz Biotechnology), mouse monoclonal Bcl2 (1 : 300; Santa Cruz Biotechnology), and mouse monoclonal cytochrome c (1 : 300; Santa Cruz Biotechnology). The immune complexes were visualized using the enhanced chemiluminescence (ECL) method.

For Western blotting, proteins obtained were subjected to SDS-PAGE on 12.5% gel. The proteins were then transferred to polyvinylidene fluoride membranes (PVDF) [Thermo Fisher Scientific, U.S.A]. Subsequently, the membrane was blotted with different antibodies like mouse monoclonal cytochrome c (1 : 300; Santa Cruz Biotechnology) and mouse monoclonal beta actin (1 : 1000; Santa Cruz Biotechnology). The immune complexes were visualized using enhanced chemiluminescence (ECL) method.