Mini trans blot electrophoretic transfer apparatus

Eluents from the affinity column were separated by SDS-PAGE and proteins were transferred onto a PVDF membrane (Thermo Fisher, USA) by using a mini trans-blot electrophoretic transfer apparatus (BioRad). Then, membrane was incubated in blocking PBS buffer containing 3% skim milk at RT for 2 h with shaking, and incubated overnight at 4°C with anti-His antibodies (1/500 dilution, monoclonal antibody, mouse IgG1, Thermo Fisher). The membrane was incubated with the secondary antibody (1/3,000 dilution, anti-Mouse IgG (H+L) conjugated with alkaline phosphatase, Bio-Rad) at 37°C for 1 h with shaking. After washing with PBST (PBS containing 0.1% (v/v) Tween-20), alkaline phosphatase substrate (Bio-Rad) was added to develop color (RT, 5 min).

Cell lysates were extracted from P10 LNC transfected with Ad-Pax6 GFP or Ad-GFP on day 4 by cold lysis buffer containing immunoprecipitation assay buffer, protease inhibitor cocktail (100x) and 1 mM phenylmethylsulfonyl fluoride.(Sigma-Aldrich, St. Louis, MO) Total protein of the cell lysate was measured and normalized by the BCA assay (Pierce, Rockford, IL) and 5 µg of protein lysate was loaded on a 4–15% (w/v) gradient sodium dodecyl sulfate-polyacrylamide gel and transferred to nitrocellulose membrane using mini Trans-blot electrophoretic transfer apparatus (Bio-Rad, Hercules, CA). Each membrane was blocked with 5% (W/V) fat-free milk in 50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, and 0.05% Tween-20 for 1 h before incubation with specific primary antibodies in 5% (W/V) fat-free milk overnight at 4 °C follow by their respective horseradish peroxidase-conjugated secondary antibodies using antibody against Histone 3 and β-actin as the loading control. Detailed information about primary and secondary antibodies and agents used is listed in Supplementary Table S4. The immunoreactive bands were detected by Western Lightning Chemiluminescence (PerkinElmer, Waltham, MA) using an ImageQuant LAS 4000 digital imaging system (GE Healthcare Piscataway, NJ).