Rabbit monoclonal antibody against gapdh

Protein was extracted using a Total Protein Extraction kit (KeyGEN Biotech, Nanjing, China), and the protein concentration was quantified using a BCA Protein Assay kit (KeyGEN Biotech). Each sample containing an equivalent amount of protein (20 µg) was separated by 10% SDS‐PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk powder for 1 hour at room temperature, the PVDF membranes were incubated overnight at 4°C with a rabbit antibody against E2F3 (1:1000; Affinity Biosciences, New Jersey, USA) and a rabbit monoclonal antibody against GAPDH (1:1000) (Beyotime Biotechnology, Beijing, China). Then, the membranes were washed three times with TBS‐T buffer, which was followed by incubation with a goat anti‐rabbit secondary antibody (1:1000; Beyotime Biotechnology) for 1 hour at room temperature. Immunoreactive proteins were detected using an ECL Reagent (Affinity) and an automatic chemiluminescence image analysis system (Tanon 5200, Shanghai, China).

A protein extraction kit (KeyGEN Biotech, Nanjing, China) was used to extract total proteins from colon cancer cells. Then, protein concentrations were quantified using a BCA Protein Assay kit (KeyGEN Biotech, Nanjing, China). After boiling for 5 min with loading buffer, each sample containing an equivalent amount of protein (20 μg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk powder for 1 h at room temperature, the PVDF membranes were incubated overnight at 4°C with a rabbit anti-TNK2 antibody (1:1,000) (Affinity, USA) and a rabbit monoclonal antibody against GAPDH (1:5,000) (Beyotime Biotechnology, Beijing, China). Then, the membranes were washed three times with TBS-T buffer, which was followed by incubation with a goat anti-rabbit secondary antibody (1:5,000, Beyotime Biotechnology) for one h at room temperature. Immunoreactive proteins were detected using an electrochemiluminescence (ECL) Reagent (Affinity, USA) and an automatic chemiluminescence image analysis system (Tanon 5200).