Western Blot Analysis of p63 Expression

Lysates from luciferase assay and ChIP were diluted in 2× loading buffer (Biorad, Hercules, CA, USA) with β-mercaptoethanol, denatured for 5 min at 95 °C and separated on a 10% SDS-page gel. After transferring to Hybond ECL nitrocellulose membrane (GE Healthcare, Little Chalfont, UK), the membrane was blocked in PBS with 0.05% Tween-20 (PBST) containing 3% non-fat dried milk for 1 h at RT, after which anti-p63 (Santa Cruz Biotechnology) was incubated for 2 hours at RT in PBST containing 5% BSA. After incubation with peroxidase labeled secondary antibody rabbit anti-mouse (Dako, Glostrup, Denmark) bands were visualized with ECL+ (GE Healthcare). Positive control lysates for endogenous p63 were obtained by pBABE-LIC-ΔNp63α transfected 293T cells. Briefly, ΔNp63α was PCR amplified from pcDNA3.1-ΔNp63α and cloned into pBABE-LIC vector (obtained from Dr. Priyamvada Rai, University of Miami, USA) as described [44 (link)].