Western Blot Analysis of PAX6 and GAPDH

Cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma) containing the protease inhibitor cocktail (Sigma) to isolate total protein, and the proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. Following blocking with 5% skim milk for 2 h, the membranes were incubated with anti-PAX6 (1:1000, ab195045, Abcam) and anti-GAPDH antibodies (1:2000, ab8245, Abcam) at 4°C overnight. After washing, the membranes were incubated with the secondary antibody (Abcam) conjugated with horseradish peroxidase for 2 h at room temperature. Enhanced chemiluminescence (ECL) substrate (Bio-Rad) was used to visualize the protein bands [22 (link)].