Western blot analysis of Bcl-2 and Bax expression

Oxidative stress ultimately causes cell apoptosis, and thus apoptosis factors were studied to determine whether LBPs could inhibit apoptosis caused by oxidative stress. The typical index of the Bcl class was selected, including the inhibitor of apoptosis protein Bcl-2 and the pro-apoptotic protein Bax, which were used to indirectly assess whether LBPs prevented apoptosis. Total protein was extracted from cells using the protein extraction kit, and quantified using the BCA kit, according to the manufacturer's instructions. Proteins (80 µg) were separated on 12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes. Membranes were then blocked (5% non-fat dry milk in PBS plus 0.1% Tween-20; room temperature; 2 h), incubated with primary antibody (4˚C; overnight) and second antibody (room temperature; 2 h) and then quantified. The primary antibodies used were anti-Bcl-2, diluted in 1% bovine serum albumin (BSA; Thermo Fisher Scientific, Inc.) to 1:200, anti-Bax, diluted in 1% BSA to 1:3,000, and anti-β-actin, diluted in 1% BSA to 1:500; the secondary antibodies were HRP-goat anti-rat IgG and HRP-goat anti-rabbit IgG, diluted in 1% BSA to 1:5,000. Bands were quantified following detection with the ECL kit using Image Lab 5.0 (Bio-Rad Laboratories, Inc.).