Mutation of T222/226 and S230 alters C/EBPα activity in preadipocytes. A) 3T3L1 preadipocytes were transfected with 2 μg of C/EBP-Luc reporter along with 200 ng of pcDNA6, Flag-C/EBPα, AA, AAA, or S230A as described in the material and methods. Cells were incubated with insulin (l00 nM) or CHIR99021 (CT, l0 μM) for 20 hr prior to harvest for luciferase assay. Results are relative luciferase activity and are average ± SEM (n = 6) from two independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.005 compared with control. B) 3T3L1 preadipocytes were transfected with 200 ng of pcDNA6, Flag-C/EBPα wild type, or Flag-C/EBPα S230A, ± 2 μM CHIR99021 (CT). Cells were lysed, the recombinant protein immunoprecipitated using anti-FLAG, and visualised using anti-FLAG or anti-phospho222/226. A representative experiment is shown in the upper panel, while a quantification of three such experiments is given in the lower panel.