Figure 4

C/EBPα phosphorylation in vitro. (A) Recombinant Flag-C/EBPα (6 pmol) was generated by scission of the GST-C/EBPα fusion protein and incubated with either recombinant GSK3β (2 U/ml) or p42 MAPK (10 U/ml) and [γ-32P]-ATP for 1, 10, 30, and 60 min. The reaction was stopped and separated by SDS-PAGE. (B) The stoichiometry of Flag-C/EBPα phosphorylation in (A) by either MAPK or GSK3β is quantified and presented. (C) GST-C/EBPα (7.5 pmol) was incubated with recombinant GSK3β (2 U/ml) and ATP for 1, 10, 30, and 60 min. The reaction was stopped and separated by SDS-PAGE and subjected to Western Blot analysis for phospho-T222/226. The equivalent stoichiometry of phosphorylation (mol/mol) calculated in A is indicated above each band.