Anti bcl 2 antibodies

The cells were harvested 48 h after transfection and lysed with RIPA buffer (ThermoFisher, USA). After centrifugation (10,000 g at 4 °C for 10 min), the concentration of the protein in the resulting supernatant was determined using a BCA Protein Assay Reagent kit (Pierce, USA). Afterward, 20 µg protein samples were resolved on a 10% SDS-PAGE gel before being transferred to PVDF membranes. Each membrane was subsequently blocked with 5% skim milk powder at room temperature for 30 min. Next, the membranes were exposed to diluted specific primary antibodies at 4 °C overnight. Thereafter, horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibodies (Sigma-Aldrich, USA) were added to the membranes at room temperature for 3 h. Finally, a Pierce ECL Western Blotting Substrate was utilized for the visualization of the bands and the same were photographed on a GelDoc XR+ system (Bio-Rad, USA). The antibodies used are as follows: anti-Bcl-2 antibodies (Cat#: 13–8800; 1:10,000; ThermoFisher, USA), anti-Bax antibodies (Cat#: MA5–14,003; 1:10,000; ThermoFisher, USA), anti-IMPA2 antibodies (Cat#: ab256410;1:10,000; Abcam, USA), anti-GAPDH antibodies (Cat#: ab99252; 1:10,000; Abcam, USA), anti-mouse HRP secondary antibody (Cat#:31,430,1:1000, Invitrogen, USA), and anti-goat HRP secondary antibody (Cat#:31,402,1:1000, Invitrogen, USA).

Cells were lysed using radioimmunoprecipitation assay buffer (Beyotime, China) and then quantitatively assessed using a bicinchoninic acid protein assay kit (Pierce, USA) and NanoDrop ND 2000 c Spectrophotometer (Thermo Scientific, USA) as per the manufacturer’s instructions. Equal amounts of denatured proteins were loaded onto 10% sulfate-polyacrylamide gel and separated via electrophoresis run at 75 to 175 V. The proteins in the gels were transferred onto polyvinylidene fluoride membranes using a wet transfer system and blocked with 5% milk in TBS at 25°C for 1 h. Membranes were continuously exposed to TBST containing anti-CKS1B antibodies (Cat#:C0748, Sigma-Aldrich, USA), anti-Bax antibodies (Cat #33-6400, 1:1000, Thermo Fisher Scientific, USA), anti-Bcl-2 antibodies (Cat #MA5-11,757, 1:1000, Thermo Fisher Scientific, USA), or anti-GAPDH antibodies (Cat #MA1-16,757,1:1000, Thermo Fisher Scientific, USA) at 4°C. Antibody-bound proteins were probed using an anti-mouse/goat HRP secondary antibody (Cat #31,430 or Cat #31,402, 1:1000, Invitrogen, USA) at room temperature for 1 h. Chemiluminescent detection (Beyotime, China) was used to visualize the target proteins, and the intensity of protein signals was estimated using Quantity One 4.1 (Bio-Rad Laboratories, USA) [22 (link)].