Western Blot Analysis of Protein Expression

Whole-cell extracts were prepared with a nuclear extraction TransAm Kit (Active Motif, Carlsbad, CA, USA) and Western blotting was carried out as previously described.21 (link),22 (link) Briefly, samples were subjected to Western blot analysis (30–40 μg) in a NuPAGE 10% Bis-Tris Gel (Thermo Fisher Scientific). Blots were blocked with 5% nonfat dry milk in Tris-buffered saline, 0.1% Tween 20 for 1 hour at room temperature (RT), and then the blots were treated with a 1:200 dilution of primary antibody for 2 hours at RT. Blots were then incubated with secondary antibody (1:3,000 dilution) for 1 hour at RT and washed seven times with Tris-buffered saline, 0.1% Tween 20. Immunoreactive bands were visualized using an ECL Plus Detection Kit (Amersham, Pharmacia Biotech, Arlington Heights, IL, USA). Membranes were stripped and reprobed for β-actin, which was used as a protein loading control.