Anti cdk1

After treating with 50μM 7,8-benzoflavone for 48 hours, protein extraction was conducted utilizing RIPA buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors in HT-29 and HCT-116 cell lines. The Western blotting procedures adhered to established protocols as delineated in prior publications 30 (link). The primary antibodies, anti-CDK1, anti-IL1RN and anti-GAPDH, were procured from Zenbio, China.

Proteins were analyzed by western blotting according to standard methods. Proteins were extracted from cells using RIPA buffer (Beyotime, China) containing a mixture of protease and phosphatase inhibitors and quantified using an Enhanced BCA Protein Assay Kit (Beyotime, China). Protein samples (30  μg/lane) were loaded onto 10% separating gel and blotted onto a PVDF membrane (Millipore, Billerica, MA, United States). After blocking with 5% skimmed milk powder for 1 h, the membranes were incubated overnight with the following primary antibodies at 4°C: anti-CDK1(Zen-BioScience, China), anti-survivin (Zen-BioScience, China), anti-MMP2 (Zen-BioScience, China) and anti-GAPDH (Zen-BioScience, China). Membranes were then washed and incubated with HRP-linked secondary antibody for 1 h at 37°C. Finally, the samples from three independent experiments were detected using the ECL reagent (Thermo Fisher, United States).