Bicinchoninic acid bca protein concentration assay kit

Protein extraction was performed on ice, and the reaction solution was pre-cooled with ice. The tissues or cells were washed with cold phosphate-buffered saline (PBS), lysed with radioimmunoprecipitation assay (RIPA) (Beyotime Institute of Biotechnology) lysis buffer, centrifuged (at 10,000 x g for 15 min at 4̊C), discarded, and resuspended in a cell lysate containing phosphatase inhibitor or protease inhibitor (Beyotime, Shanghai, China). Bicinchoninic acid (BCA) protein concentration assay kit (Beyotime Institute of Biotechnology) was used for the determination of protein quality. Then, the protein was added to the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membranes (EMD Millipore). After 5% milk blocking, membranes were coated with primary antibodies against: Collagen II (ab34712), ATF4 (ab31390), CHOP (ab240220), cleaved caspase-9 (ab2324), and β-actin (as loading control, ab179467) overnight at 4˚C. All the antibodies were purchased from Abcam. Membranes were then incubated with secondary antibody for 1 h at room temperature. Chemiluminescent ECL substrate (Beyotime Institute of Biotechnology) was used to expose the band.

Nasopharyngeal carcinoma HK-1 cell line was purchased from ATCC (Washington D.C., USA) and preserved by our studying team; fetal bovine serum, RPMI-1640, and Opti-MEM medium were purchased from Gibco (Beijing, China); dimethyl sulfoxide (DMSO) was purchased from Sigma (Shanghai, China); phosphate-buffered saline (PBS) powder was purchased from Wuhan PhD Bioengineering Co., Ltd (Wuhan, China); Lipofectamine 2000 transfection reagent was purchased from Invitrogen (Wuhan, China); Radio-Immunoprecipitation Assay (RIPA) lysate, phenylmethylsulfonyl fluoride, bicinchoninic acid (BCA) protein concentration assay kit, SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) gel rapid preparation kit (P0012 AC), PTEN, PI3K, p-Akt, FoxO3a first antibody and internal reference ACTB antibody, and goat antirabbit fluorescent second antibody were purchased from Beyotime Biotechnology.