Figure 5

S230 phosphorylation does not prime for T226 phosphorylation by GSK3. (A) AD293 cells were transfected with 10 μg of Flag-C/EBPα, Flag-C/EBPα-AA, Flag-C/EBPα-AAA or Flag-C/EBPα-S230A prior to 24 h incubation with 2 μM CHIR99021 (CT), lysis and Western Blot analysis. A representative immunoblot of two such experiments is given. (B) AD293 cells were transfected with 10 μg of Flag-C/EBPα or Flag-C/EBPα-S230A, then serum starved overnight prior to a 2 h incubation with CHIR99021 (2 μM) or 10%(v/v) FBS. Cell lysates were then subjected to Western Blotting with the antibodies as labelled. A representative immunoblot of two such experiments is given.