FIG. 4.

Phosphorylation of Sox9 in intact cells. (A) Endogenous Sox9 is phosphorylated in RCS cells. RCS cells in culture were incubated with ³²P-labeled inorganic phosphate for 4 h. Sox9 was immunoprecipitated from cell lysates with SOX9 antibody (Ab) and visualized by autoradiography after SDS-PAGE (lane SOX9 Ab). A unique ³²P-labeled band migrated with the characteristic 68-kDa molecular mass of SOX9. The specificity of the immunoprecipitation was assessed with SOX9 preimmune (Pre-immu.) serum. (B) PKA-Cα phosphorylates SOX9 at sites 1 and 2 in COS-7 cells. COS-7 cells were transfected with plasmids expressing wild-type SOX9 (wt), double-mutant SOX9 (m1+2), and PKA-Cα, as indicated. Cells were incubated with ³²P, and lysates were immunoprecipitated with SOX9 antibody. Autoradiographs of phosphorylated SOX9 proteins recovered after immunoprecipitation and SDS-PAGE are shown in the top panel. PKA increased the level of phosphorylation of wild-type SOX9 but not that of m1+2 mutant SOX9. A Western blot showing the amount of SOX9 protein in each sample after immunoprecipitation is presented in the bottom panel.