Beyoecl star ultrasensitive ecl chemiluminescence kit

Lysis buffer (0.5 ml) was added and thoroughly mixed with 100 mg of the tissues cut into small pieces. After the mixture was allowed to stand for 10 min at 4°C, 1 ml of the extraction reagent was added and mixed well with the solution. Then, the resulting solution was left to stand for another 10 min at 4°C. Subsequently, it was centrifuged at 10,000 g at 4°C for 10 min to obtain two phases of liquid separated by a protein layer. The liquid was removed, and the proteins were dried at 4°C. Then, 2% SDS was used to dissolve the proteins, and the BCA assay was performed for protein quantification. The protein solution was fully mixed with a loading buffer at a ratio of 3:1 and then placed in a water bath at 95°C for 10 min (denaturation). Wells were loaded with 30 μg of proteins, and SDS‐PAGE was performed at 80 V for 2 hr. Membranes were transferred at 70 V for another 2 hr. After the membranes were mounted with 5% skim milk/TBST for 1 hr, the proteins were incubated overnight at 4°C with AKT, p‐AKT, caspase‐3, and caspase‐9 (1:500) and then placed onto a thermostatic shaking table for secondary antibody incubation (1:5,000) at room temperature for 1 hr. A BeyoECL Star Ultrasensitive ECL chemiluminescence kit (Beyotime) was used for development, and a Bio‐Rad gel imaging system was employed to capture images. Image analysis was performed using Image Lab 5.0.

After cells treated with different cell culture medium for 48 h, cells were treated with 10 µM MG132 (MedChem Express, HY-13259) for 6 h before harvesting. Then cells were collected and whole proteins were withdrawn using a mixture of 100 µL of lysis buffer, consisted of 1 µL of protease inhibitor (100 ×), 1 µL of phosphatase inhibitor (100 ×) and 1 µL of benzenesulfonyl fluoride (100 ×). Protein samples were denatured in 10% SDS sample buffer (Beyotime Biotechnology, China) and separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) under denaturing conditions. After protein was dissociated, it was electroblotted onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA), blocked with 5% skim milk powder, and then and incubated with diluted primary antibody overnight at 4 ℃. Then the hybridization bands were incubated with horseradish peroxidase-conjugated secondary antibodies (Zhongshan, Beijing, China) at 37 ℃ for 1 h. Protein concentrations were measured using the commercially available BeyoECL Star ultra-sensitive ECL chemiluminescence kit (Beyotime Biotechnology, China). Protein expression was quantified with ImageJ software (NIH, Bethesda, MD, USA). Primary antibodies were β-actin (1:1000, TA-09, Zhongshan, Beijing, China) and P53 antibody (1:1000, 2524S, CST, USA).