Ripa buffer

RIPA buffer supplemented with protease and phosphatase inhibitors (TargetMol, America) was used to lyse the cells. The protein supernatant was then collected and preserved at −80°C. The BCA (Beyotime, China) method was used to detect protein concentrations. The sample proteins were first heated at 99°C for 20 min, then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Servicebio, China) and transferred to polyvinylidene fluoride membranes. After blocking with 5% non-fat milk for 90 min, the membranes were incubated with primary antibodies at 4°C overnight, then washed and incubated with secondary antibodies for another 2 h at room temperature. An enhanced chemiluminescence reagent (Servicebio, China) was used to image the protein bands. The primary antibodies used in this study were PARP1 (1:1,000, CST, America) and GAPDH (1:10,000, Proteintech, USA). The secondary antibodies were purchased from HuaBio, China.

Cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors (TargetMol, America), and the protein supernatant was stored at −80°C. The protein concentration was detected by the BCA (Beyotime, China) method. After reduction by heating at 99°C for 20 min, the sample proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Servicebio, China) and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 90 min followed by incubation with the primary antibody at 4°C overnight and then with the secondary antibody for 2 h at room temperature. Finally, enhanced chemiluminescence (ECL) reagent (EpiZyme, China) was applied to visualize the protein bands. The primary antibodies involved in this work include EGFR (1:1000, CST, America), p-EGFR (1:1000, CST, America), AKT (1:1000, CST, America), p-AKT (1:1000, CST, America), and GAPDH (1:1000, Abclonal, China). Secondary antibodies were purchased from ABclonal, China.

Colorectal cancer specimens and colorectal cancer cells were lysed with RIPA buffer (TargetMol, USA) containing protease inhibitor and phosphatase inhibitor. The supernatant was collected after centrifugation at 4°C for 30 min. After the protein was quantified and denatured, the denatured protein was separated by SDS-PAGE electrophoresis and then transferred to a PVDF membrane. After blocking the membrane with 3% bovine serum albumin for 1 h, primary antibody was added and incubated overnight at 4°C. After removal of the primary antibody, the membrane was washed three times with TBST and incubated with secondary antibody at room temperature for 1 h. Immune complexes were detected by an enhanced chemiluminescence system (Life Tec, USA), and protein bands were analyzed and quantified using ImageJ software (version 11). The primary antibodies were as follows: caspase 3 (Abclonal, China), caspase 9 (Abclonal, China), cleaved caspase 3 (CST, USA), CYP24A1 (CST, America), cleaved caspase 9 (Abclonal, China), γh2A (Abclonal, China), Ki67 (Abclonal, China) and GAPDH (Abclonal, China). The secondary antibody was purchased from Abclonal (China).