*2.4. Western Immunoblotting*

The levels of proteins involved in apoptotic signaling—Bax, Bcl-2, and cleaved caspase-3—were determined via Western blotting. Total protein (20 µg) from homogenized LV tissues was denatured at 95 ◦C for 5 min. The samples were loaded onto 10–12% SDS–polyacrylamide gels and resolved by electrophoresis at 110 V for 2 h. The proteins were electro-transferred onto a nitrocellulose membrane (Pall Corporation, Port Washington, NY, USA) for 1 h at 260 mA. Thereafter, the membranes were stained with Ponceau S (Sigma-Aldrich, St. Louis, MO, USA) to ensure equal loading and protein transfer of all samples. The membranes were blocked with 5% skim milk in TBS buffer with 0.1% Tween-20 (TBST) for 2 h at room temperature, washed thrice for 10 min with TBST, and incubated at 4 ◦C for 12 h, with the following primary antibodies (diluted in TBS): anti-Bax (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Bcl-2 (1:1000; Santa Cruz Biotechnology), and anti-cleaved caspase-3 antibodies (1:500; Cell Signaling Technology, Beverly, MA, USA). The membranes were then washed thrice for 10 min with TBST. Thereafter, the membranes were slowly incubated on a shaker for 1 h with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:3000 in TBST; Santa Cruz) at room temperature. After washing with TBST, protein bands were developed using an enhanced chemiluminescence detection kit (Thermo Scientific, Waltham, MA, USA). Relative protein quantification was performed by densitometry analysis using the Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA).
