Quantitative Western Blot Analysis

Western blotting was carried out as previously described [26 (link)]. Briefly, RIPA buffer (R0278, Sigma-Aldrich) with a protease inhibitor cocktail (complete Tablets Mini, Roche, Basel, Switzerland) was used to isolate total protein from cells. A colorimetric test (PierceTM BCA Protein Assay Kit, Thermo Fisher Scientific) was used to determine the total protein concentration. SDS-PAGE was used to separate 10 µg of total protein under reducing conditions, and immunoblot analyses were carried out with antibodies (Supplementary Table S1) against PAX6 (clone#poly901301—1:1000; clone#D3A9V—1:1000) and GAPDH (1:50,000), followed by horseradish peroxidase-labeled anti-mouse or rabbit IgG (Jackson ImmunoResearch Europe, Ely, UK). Enhanced chemiluminescence Western blot detection reagent (GE Healthcare, München, Germany) and FUSION FX imager/fusion software (Vilber Lourmat, Collégien, France) were used to visualize protein bands.