Ecl chromogenic solution

Cells at a density of 3 × 10⁵ cells/well were incubated overnight in a 6-well plate. After incubation with different drugs for 24 h, RIPA lysis buffer containing a protease inhibitor and phenylmethylsulfonyl fluoride (Beyotime BioTECH, Shanghai, China) was used for cell lysis on ice for 30 min. Protein concentration was determined using a BCA assay kit (Beyotime BioTECH). Protein samples were added to a protein-loading buffer (Beyotime BioTECH), boiled at 100°C for 8 min, separated using polyacrylamide gel electrophoresis (Epizyme, Shanghai, China), and transferred to a polyvinylidene fluoride membrane (Millipore, Massachusetts, USA). The membrane was sealed with a rapid blocking solution (Beyotime BioTECH) for 30 min at room temperature and then incubated with primary antibodies (diluted at 1 : 1000) at 4°C overnight. Antibodies against HIF-1α, VEGF, AKT, p-AKT, ERK, and p-ERK were purchased from CST (Boston, USA). Secondary antibodies labeled with horseradish peroxidase (KeyGEN BioTECH) were then incubated (diluted at 1 : 1500) at room temperature for 1 h. Finally, an ECL chromogenic solution (Beyotime BioTECH) was prepared and dropped on the membrane surface, which was exposed and photographed.

The cells were lysed by cell lysis buffer for Western and IP (Beyotime, Shanghai, China, P0013) at 4 °C for half an hour, and the protein sample from the supernatant was obtained after centrifuging the cell lysate. The protein sample was prepared with 5× loading buffer and centrifuged after a 100 °C water bath. Then the protein sample was separated with 12.5% PAGE Gel. After electrophoresis, the gel was transferred to a polyvinylidene difluoride (PVDF) membrane (Roche, Basel, Switzerland, 65310600) on ice for 90 min at a 100 V voltage by Power Pac (Bio-Rad, Hercules, USA). After that, the PVDF membrane was sealed with 5% skim milk powder compounded by TBST for 2 h at room temperature. Next, the PVDF membrane was incubated with the corresponding primary antibody and secondary antibody at 4 °C for 4 h and 1 h, respectively. Finally, the membrane was treated with ECL Chromogenic solution (Beyotime, Shanghai, China, P0018M), and detected by using a Tanon 5500 chemiluminescence instrument.