RA decreases cyclin H-cdk7 expression and inhibits RARα phosphorylation. (A) SCC4 and SCC25 cells were treated with vehicle or 1 μM RA (+RA) for 16 hours as described in Methods. Cyclin H and cdk7 expression was determined by western blot. Blots were stripped and incubated with anti-β-actin antibody to ensure equal amounts of protein in each lane. (B) RA inhibits RARα phosphorylation. RARα was immunoprecipitated (IP RARα) from lysates of vehicle and RA treated SCC4 and SCC25 cells as described in Methods. Blots were incubated with anti-phosphoserine antibodies (anti-pSer) followed by stripping and probing with anti-RARα antibodies to ensure equal relative amounts of immunoprecipitated protein in each lane. To determine the specificity of this assay, SCC25 cells were treated with vehicle or the selective MAPK inhibitors PD98059 (50 μM) or SB203580 (10 μM) for up to 24 hours. RARα protein was immunoprecipitated as described above. These experiments were performed three times with similar results. Representative blots are shown.