Ab129192

Collected cells, which were treated with 2.5 and 5 mg/mL of TM for 24 h, and collected tumor tissues were lysed by RIPA buffer (Sigma-Aldrich, USA) containing 1% protease inhibitor cocktail (Sigma-Aldrich, USA) and 2% phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich, USA). Protein concentrations were determined using Bradford method. 40 μg of proteins was separated by 10–12% SDS-PAGE gels and then transferred electrophoretically onto PVDF membranes. The transferred membranes were blocked in 5% bull serum albumin (BSA) for 4 h and then blotted with the following primary antibodies at 4°C overnight at dilution of 1 : 1000: cleaved poly(ADP-ribose) polymerase (cleaved-PARP) (ab32064), Bad (ab129192), Bax (ab7977), B-cell lymphoma-2 (Bcl-2) (ab32124), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245) (Abcam, Cambridge, MA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies diluted at 1 : 2000 (Santa Cruz, USA). ECL detection kits (Millipore, USA) were applied to detect chemiluminescence of blots, and the intensity was quantified by scanning densitometry using Image J (NIH, Bethesda, MD).

Total proteins were extracted with RIPA buffer containing protease inhibitor and quantified with a BCA protein analysis kit (Beyotime, Shanghai, China). Next, 20 μg of protein was separated by 10% SDS-PAGE (Thermo Fisher Scientific, Inc., Waltham, USA) and transferred to PVDF membranes. The membranes were incubated in 5% non-fat milk at room temperature for 2 h. Afterward, the membranes were incubated at 4° C overnight in dark with the following primary antibodies: anti-ErbB3 (1:1000; ab32121; Abcam, Shanghai, China), anti-p-AKT (1:1000; ab38449), anti-AKT (1:1000; ab179463; Abcam), anti-Bad (1:2000; ab32445; Abcam), anti-p-Bad (1:5000; ab129192; Abcam), anti-cleaved Caspase 3 (1:500; ab32042; Abcam), anti-Bcl-2 (1:1000; ab32124; Abcam), and anti-GAPDH (1:2500; ab9485; Abcam). Next, after washing with Tris/Tween-20 for 3 times, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000; ab7090; Abcam) at room temperature for another 2 h. Finally, the proteins were visualized by BeyoECL Plus Kit (Beyotime) and quantified with the ImageJ software (version 1.8.0, National Institutes of Health, USA).