Bax ab32503

Cells were harvested and suspended in RIPA lysis buffer. Protein concentrations were detected using BCA protein assay kit (Beyotime, China). Equal amounts of protein was resolved on a 10% sodium dodecyl sulfate polyacrylamide gel (SDS‐PAGE) and transferred to a PVDF membrane (Millipore, USA). This was followed by blocking with 5% BSA, and the PVDF membranes were incubated with primary antibody overnight at 4°C. The primary antibodies included Bcl‐2 (#15071), E‐cadherin (#14472), Vimentin (#5471), β‐catenin (#8480), STAT3 (#9139), p‐STAT3 (#9145) (Cell Signaling Technology, MA, USA); Bax (ab32503), HIF‐1α (ab51608), VEGFA (ab52917) (Abcam, Cambridge, MA, USA) and GAPDH (G9545, Sigma, St Louis, MO, USA). Membranes were washed with TBST and incubated with HRP‐ conjugated secondary antibody. Proteins were visualized by ECL Western blot analysis system.

Total cellular proteins were extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, P.R. China) supplemented with protease inhibitors (Roche, Guangzhou, P.R. China). The proteins were quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). Whole-protein samples were separated using a Bio-Rad Bis-Tris Gel system (Bio-Rad) according to the manufacturer’s instructions and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in 5% nonfat milk for 2 h at room temperature and then incubated overnight at 4°C with the following primary antibodies diluted 1:1,000 in blocking buffer: Bcl-2 (ab32124; Abcam, Cambridge, MA, USA), Bax (ab32503; Abcam), caspase 3 (3CSP03; Santa Cruz Biotechnology), TPM1 (ab55915; Abcam), and anti-GAPDH antibody (ab8245; 1:100 dilution; Abcam). After washing, the membranes were incubated for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000 dilution; ERP3312, ab197034; Sigma-Aldrich). After washing, the membranes were transferred into the Bio-Rad ChemiDoc™ XRS system, and 200 μl of Immobilon Western Chemiluminescent HRP Substrate (Millipore, Boston, MA, USA) was added to cover the surface. The signals were captured, and the intensity of the bands was quantified using Image Lab™ Software (Bio-Rad, Shanghai, P.R. China).

Total protein from THCA cells was extracted with the help of RIPA lysis buffer (Thermo Fisher Scientific) having protease inhibitor cocktail (Roche, Basel, Switzerland). The quantity of individual protein samples was evaluated availing a BCA test kit (Thermo Fisher Scientific). Through a 12 % SDS-PAGE, protein samples (20 μg) were separated at 150 V for 1.5 h and then transferred onto PVFD membranes (Millipore). Following blocking these with 5 % non-fat milk, the membranes were treated with primary antibodies at 4 °C overnight. The following antibodies were used in this study at a dilution of 1:1000 and were acquired from abcam, Cambridge, UK - Bcl-2 (ab32124), Bax (ab32503), EVA1A (ab216043), and GAPDH (ab9485). The membranes were then treated with hrp-conjugated goat anti-rabbit IgG secondary antibody (abcam, 1:10,000). Finally, each membrane was visualized using a chemical immunogenicity system (GE Healthcare, Chicago, IL, USA), and ImageJ software (NIH, Bethesda, MD, USA) was employed for quantitation of the intensities of the protein bands.