Cw0043s

Collecting cells and using the radio immunoprecipitation assay (RIPA) buffer (P0013B, Beyotime, Shanghai, China) in the presence of a phenylmethyl sulfonylfluoride (#8553, CST). Protein concentration were determined by the BCA Protein Assay Kit (#P0009, Beyotime, China). Equivalent amounts of the proteins (40 μg/line) were separated on Tris-Tricine Ready Gel (#1611210, Bio-Rad, Hercules, Calif, USA) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for nitrocellulose membrane polyvinylidene fluoride (PVDF) (ISEQ00010, Merck Millipore, Massachusetts, USA) blotting. The blotted membranes were blocked with 5% skim milk (#P0216-300g, Beyotime, Shanghai, China) for 1 hour at ambient temperature and incubated with primary antibodies overnight at 4^◦C. They were washed with tris-buffered saline Tween-20 (TBST) (CW0043S, CWBIO, Jiangsu, China), and the immunoreactive bands were visualized by enhanced chemiluminescence reagent. All bands were analyzed by NIH image 1.62. All experiments were conducted in triplicate.

HCT116p53^+/+ and HCT116p53^–/– cells were seeded in 6 cm dish at a density of 6 × 10⁵ cells per well. Incubated overnight, add different treatment group media (control, 5-Fu, β-elemene, 5-Fu + β-elemene) for 24 h. Cells were harvested and lysed using the RIPA buffer (P0013B, Beyotime) in the presence of a phenylmethyl sulfonylfluoride (PMSF) (#8553, CST). Protein concentration was determined using the BCA Protein Assay Kit (P0009, Beyotime). Equivalent amounts of protein were resolved and mixed with 5× SDS-PAGE protein sample buffer (P0015, Beyotime), electrophoresed in SDS-PAGE, transferred to PVDF membranes (Merck Millipore, Billerica, MA, United States). The blotted membranes were blocked with 5% skim milk for 1 h and incubated with primary antibodies overnight at 4°C. Day 2, washed with TBST (CW0043S, CWBIO), then incubated with suitable HRP-conjugated second antibodies and subjected to enhanced chemiluminescent staining using an ECL detection system (Bio-Rad). All experiments were conducted in triplicate.