Ripa lysis buffer

A protein extraction RIPA lysis buffer (Elpis Biotech) containing a Halt^™ Protease Inhibitor Cocktail (Thermo Fisher Scientific, Inc.) was used to lyse the treated cells. Thereafter, Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Inc.) was used to quantify the extracted protein. Equal amounts of proteins (50 µg/lane) were separated by 12.5% SDS-PAGE gel and transferred to a PVDF membrane (MilliporeSigma). The membranes were then blocked with 5% skimmed milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) at room temperature for 2 h. The primary antibodies used were anti-Bax (1:500), anti-Bcl-2 (1:500), anti-PARP (1:500), anti-caspase-3 (1:500), anti-caspase-9 (1:500), and β-actin (1:1,000). The membranes were incubated with primary antibodies diluted in TBST overnight at 4˚C, washed three times with TBST. Finally, a western blotting detection kit (ECL Western blotting substrate kit; Abcam) was used to observe the protein bands after treatment with a horseradish peroxidase-linked secondary antibody (1:1,000, Advansta) at room temperature for 1 h.

Immunoblotting was performed, as described previously with some modifications [54 (link)]. For the preparation of whole-cell lysates, cells were lysed for 30 min on ice through the addition of RIPA lysis buffer (ELPIS Biotech, EBA1149) plus protease inhibitor cocktail (Sigma, P8340) and phosphatase inhibitor cocktail (Sigma, P0044, P5726). Centrifugation was performed at 12,000 × g for 30 min at 4°C, and the supernatants were collected and used as protein extracts. Protein extracts were added to sample buffer, boiled for 5 min, and stored at – 80°C until use. Samples containing 20–40 µg of protein extract were separated on a polyacrylamide gel and transferred to a PVDF membrane (Bio-rad, 162–0177), which was blocked with 5% nonfat dry milk (Bio-rad, 170–6404) in 0.05% Tween-Tris-buffered saline (T-TBS; 20 mM Tris, 150 mM NaCl, 0.05% Tween 20 (Sigma-Aldrich, P9416), pH 7.5) for 60 min at room temperature (RT). Blots were probed overnight at 4°C with the relevant antibodies, washed, and probed again with species-specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, sc-2004, sc-2005, sc-2006). Protein signals were visualized using an enhanced chemiluminescence reagent.