Radioimmunoprecipitation assay protein lysis buffer

MDA-MB-231 cells treated with metformin (5 mM) were collected at various time points (0, 6, 16 and 24 h), washed twice with ice-cold PBS, and incubated in radioimmunoprecipitation assay protein lysis buffer (Beyotime Institute of Biotechnology) for 30 min at 4°C. The lysates were centrifuged at 13,000 × g for 10 min at 4°C. The concentrations of total lysate protein were detected by a standard Bradford assay (Bio-Rad Laboratories, Inc., San Diego, CA, USA), resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime Institute of Biotechnology), and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc.). The nitrocellulose membranes were blocked with 5% non-fat milk at room temperature for 1 h and then incubated overnight at 4°C with rabbit anti-BCL-2 (1:1,000), anti-MCL-1 (1:500), anti-BAX (1:1,000) and anti-β-actin (1:1,000) primary antibodies. After washing the membranes three times for 10 min each with Tris-buffered saline containing Tween-20, they were incubated with HRP-conjugated secondary antibodies (1:5,000 dilution). Proteins were visualized using an Enhanced Chemiluminescence-Plus kit (Nanjing KeyGen Biotech Co., Ltd.).

Western blot was performed to detect the expression and phosphorylation of ATM kinase and Chk2 in the A549 cells. The treatment schedule for Western blot tests was the same as that for cell cycle assay. At 3 hours post-irradiation, A549 cells in each group were lysed in 100 µL of radio-immunoprecipitation assay protein-lysis buffer (Beyotime Institute of Biotechnology) supplemented with 1 nM phenylmethylsulfonyl fluoride and 1 nM sodium orthovanadate. Proteins were extracted on ice for at least 30 minutes.
The concentrations of the proteins in the lysates were measured using a bicinchoninic acid protein assay kit. Lysate proteins (50 µg) were fractionated by 12% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, Waltham, MA, USA) and blocked for 1 hour in Tris-buffered saline containing 5% bovine serum albumin and 0.05% polysorbate 20 at room temperature. Blots were probed with the appropriate primary antibodies and peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies. Specific signals were detected with an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology). The images were analyzed using Adobe Photoshop CS3 (Adobe Systems Incorporated, San Jose, CA, USA).