Mouse monoclonal bcl2

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.

Proteins were separated on 4–20% mini-PROTEAN^®TGX™ pre-cast protein gels (Bio-Rad, Watford, UK; B-R) and transferred onto a polyvinylidene diflouride (PVDF) membrane using a Trans-Blot^®Turbo™ transfer pack (B-R). Following the transfer, membranes were incubated in 5% powdered milk (Marvel^®) dissolved in PBST (1× PBS containing 0.1% (v/v) Tween20) and agitated at room temperature for 1 h to prevent non-specific binding. Subsequently, membranes were treated with either rabbit polyclonal EGFP, mouse monoclonal actin (Abcam, Cambridge, UK), mouse monoclonal BCL2 (Santa Cruz, Dallas, TX, USA) or rabbit monoclonal VSV-G (Abcam) primary antibodies for 1 h. Membranes were washed 4 × 10 min with PBST and incubated with a species-specific secondary antibody conjugated with horseradish peroxidase for 1 h. To remove any background, membranes were washed 4 × 10 min with PBST. Bound proteins were visualized using Clarity™ Western ECL blotting substrates (B-R). After a 5 min incubation, the bands were imaged using a ChemiDoc™MP imaging system (B-R). The band intensities for actin (aⁱ) were used as an internal reference for each sample to calculate the amount of synthesis of either protein targets (tⁱ) EGFP or BCL2 (aⁱ/tⁱ × 100), expressed as a percentage increase.