The S77A mutant is not phosphorylated on serine in SCC25 cells. Cells were stably transfected with HA tagged RARα (HA-RAR1,2), S77A mutant (HA-S77A1,2), or G418 resistance (neo) expression vectors. HA tagged RARα proteins were immunoprecipitated from cellular lysates using anti-HA antibody (IP HA). Blots were incubated with anti-phosphoserine antibodies (anti-pSer) to determine relative levels of RARα phosphorylation on serine residues. Blots were stripped and incubated with anti-RARα antibody to confirm the identity of the anti-HA immunoprecipitates. Blots were incubated with anti-HA antibody to ensure equal amounts of immunoprecipitated protein in each lane. These experiments were performed three times using different clones with similar results. Representative blots are shown.