Ab13586

Proteins were extracted from cells of each group and their concentration was determined by BCA Kit (Biyuntian Biotech Company, China) according to instructions. The protein extracts were added with loading buffer and boiled for 10 min at 95 °C. Each well was loaded with 30 μg of samples. Then, 10% polyacrylamide gel electrophoresis (PAGE) was used to isolate proteins before polyvinylidene fluoride (PVDF) (Millipore, USA) transmembrane and 1 hr of closing with 5% bovine serum albumin (BSA) at room temperature. Later, primary antibodies BAK1 (ab104124, Abcam Company), caspase-3 (ab13586, Abcam Company), Bax (ab32503, Abcam Company), Bcl-2 (ab32124, Abcam Company), and β-actin (ab8226, Abcam Company) were added respectively for overnight incubation at 4 °C. Later, after washing with TBST (Tris-buffered saline with Tween 20) for 5 min×3 times, horseradish peroxidase- conjugated IgG secondary antibodies (Jackson Labs) were added for 1 hr of incubation at room temperature. Finally, the proteins were visualized by the chemilu -minescence reagent and analyzed Bio-rad Gel Dol EZ imager (GEL DOC EZ IMAGER, Bio-rad, California, USA). Grey value analysis of the target band was analyzed using software image J. We took β-actin as the internal reference gene.

Cells were washed with PBS and lysed on ice in RIPA buffer (V900854, Sigma) containing a cocktail of protease inhibitors (P8340, Sigma) following the manufacturers protocols. Protein concentration was measured using the BCA assay. The protein fractions were suspended in loading buffer and denatured at 100°C for 5 min. Total proteins (20 µg/lane) were separated on 12% SDS-PAGE and transferred to PVDF membranes, which were blocked in 5% fat free milk in TBST buffer (0.1% Tween-20) for 2 h at room temperature. BRCA1 levels were analyzed using a mouse monoclonal anti-BRCA1 antibody (1:1,000; ab16780; Abcam); Bcl-2, Bax, pro-caspase-3, cleaved-caspase-3, and cytochrome c levels were detected using the following mouse or rabbit monoclonal antibodies at a dilution of 1:1,000: ab117115, ab5714, ab13586, ab32042, and ab13575, respectively (Abcam). The secondary antibodies used were goat anti-rabbit antibody (1:1,000; Sc2030; Santa Cruz Biotechnology) and rabbit anti-mouse antibody (1:1,000; Sc358917; Santa Cruz Biotechnology). GAPDH was used as the endogenous control to normalize the expression levels of the proteins of interest. GAPDH levels were detected using HRP-conjugated mouse anti-GAPDH monoclonal antibody (1:5,000; HRP-60004, Proteintech). The densitometry scan of the western blotting was performed by Image Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).