C/EBPα phosphorylation in intact cells. AD293 cells were transiently transfected with Flag-C/EBPα (10 μg) prior to incubation with CHIR99021 as indicated (A). Cell lysates were then subjected to Western Blotting with the antibodies as labelled. A representative immunoblot (of 2 two separate experiments) is given. (B) AD293 cells were transfected with Flag-C/EBPα (10 μg) plus GSK3α-V5 (10 μg) or pcDNA6 (10 μg) prior to 24 h incubation with CHIR99021 (2 μM), lysis and Western Blot analysis. A representative immunoblot (of three separate experiments) is given. (C) H4IIE cells were infected with adenovirus expressing Flag-C/EBPα. After 16 h the infected cells were serum starved for 3 h prior to incubation with or without insulin (10 nM) or CHIR99021 (2 μM) for the times indicated. Cell lysates were immunoblotted with the antibodies indicated and a representative experiment is provided.