Horseradish peroxidase conjugated goat anti rabbit igg h l

Cells were lysed in ice-cold RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology). The cells lysates were centrifuged at 12,000 × g for 15 min at 4°C and the supernatants were stored at −80°C. The protein concentration was determined via BCA method. The protein extracts were fractionated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel, transferred onto PVDF membranes and blocked with 5% non-fat milk powder for 2 h at room temperature. Then the membranes were incubated overnight at 4°C with primary antibodies as follows: Anti-SAMD4B (ProteinTech Group, Inc.; 1:800; cat. no. 17723-1-AP), anti-Bax (ProteinTech Group, Inc.; 1:2,000; cat. no. 50599-2-Ig), anti-Bcl2 (Abcam; 1:2,000; cat. no. ab182858) and anti-GAPDH (ProteinTech Group, Inc.; 1:10,000; cat. no. 10494-1-AP). After washing with 1X TBST (0.1% Tween) buffer four times, the blots were incubated with horseradish peroxidase-conjugated Goat Anti-Rabbit IgG(H+L; ProteinTech Group, Inc.; 1:2,000; cat. no. SA00001-2) at 4°C for 1 h. Exposure imaging was performed using the Bio-Rad chemiluminescence imaging system (Bio-Rad Laboratories, Inc.). The blot optical density was determined by Image Lab software 5.2.1 (Bio-Rad Laboratories, Inc.).

The western blot assays were performed following previously recorded procedures.23 In short, cancer cells were lysed using RIPA buffer. The extracted proteins whose concentrations were measured using a BCA Protein Assay Kit were blended with loading buffer and then subjected to SDS/PAGE. After electroblotting to PVDF membranes, the membrane was blocked in Tris‐buffered saline with Tween‐20 (TBST) buffer containing nonfat milk. The detection of the target proteins in the membranes was carried out using rabbit anti‐CTIP (1:500 dilution, Proteintech, IL, USA) or anti‐GADPH antibody (1:200 dilution, Saierbio, Tianjin, China), and nonspecific antibodies attached to the membrane were removed after the membrane was washed with TBST buffer. Then, the membrane was incubated with horseradish peroxidase‐conjugated goat anti‐rabbit IgG (H + L) (1:2000 dilution, Proteintech, IL, USA) and washed with TBST buffer. The protein levels were determined using an enhanced chemiluminescence detection kit (Bosterbio, Pleasanton, CA, USA) and analysed using AlphaView SA software (Proteinsimple, San Jose, California, USA).