FIG. 2.

MafA serine 14 and serine 65 are major phosphorylation sites. (A) Amino acid sequence comparison of the TAD of large Maf proteins (quail MafA, chicken MafB and c-Maf, and human NRL). Conserved sequences fitting the proline-directed sequence consensus and surrounding serines 14 and 65 in MafA are highlighted. (B) Western blot analysis of WT and mutated MafA. pcDNA3-derived vectors encoding either WT, S14A, S65A, or S14A/S65A MafA were transfected into HeLa cells, and cellular extracts were analyzed by immunoprecipitation with MafA-directed serum and Western blotting with anti-HA1 antibody. (C) HeLa cells transfected with either pcDNA3-WT or pcDNA3-S14A/S65A were metabolically labeled with [³²P]orthophosphate, and cellular extracts were immunoprecipitated with MafA-directed serum. Immune complexes were analyzed by Western blotting followed by either incubation with anti-HA1 antibody (left panel) or direct autoradiography (right panel). In the lower panel, S14A/S65A MafA immunoprecipitates were treated with λ phosphatase (+) or left untreated (−) and analyzed by Western blotting with anti-HA1 antibody.