Anti rabbit igg horseradish peroxidase hrp

Total proteins were extracted from cells and protein concentrations were determined using the BCA Protein Assay kit (Takara). Proteins were separated on 12% sodium lauryl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (PVDF; Millipore, USA). After blocked with 5% non-fat skimmed milk powder at 37°C for 2 h, the membranes were incubated with primary antibodies: anti-cyclin-dependent kinase 4 (anti-CDK4) antibody (1:5000, Abcam, UK), anti-CDK6 antibody (1:5000, Abcam), anti-CCND1 antibody (1:5000, Abcam), anti-Caspase 3 antibody (1:5000, Abcam), anti-p-AKT antibody (1:500, Abcam) and GAPDH diluted at 1:2000 (Abcam) for 1 h at 37°C. The second antibody was anti-rabbit IgG-horseradish peroxidase (HRP, 1:4000; Santa Cruz, USA). Proteins were detected by enhanced chemiluminescence as described by the manufacturer (Beyotime, China).

The protein samples were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Bio‐Rad). Membranes were blocked with 10% fat‐free milk in Tris‐buffered saline containing 0.1% Tween‐20 (TBST) for 2 hr at room temperature, and then incubated with anti‐rabbit PAX6 antibody (1:1,000; catalog 42‐6600; Thermo Fisher Scientific, Waltham, MA, USA) in 2% bovine serum albumin (BSA; Amresco) at 4°C overnight. Then, after washing, the membranes were incubated with anti‐rabbit IgG‐horseradish peroxidase (HRP) at room temperature for 1 hr (1:3,000; catalog sc‐2004; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein bands were visualized using the Western Lightning Plus‐ECL kit (PerkinElmer, Akron, OH, USA). Bands were normalized to β‐actin by stripping the membranes and reprobing with mouse monoclonal β‐actin primary antibody (1:10,000; catalog A5316; Sigma‐Aldrich) and anti‐mouse IgG‐HRP secondary antibody (1:3,000; catalog sc‐2005; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Films were scanned and then analyzed with Image J software (Version 1.45 m, NIH).