C/EBPϵ is phosphorylated on Thr75 by p38 MAP kinase. In vitro–kinased GST-C/EBPϵ was treated with 6M HCl to liberate the phosphorylated residues that were resolved by 2D electrophoresis in buffers pH 1.9 and pH 3.5. (A) ERK2 and (B) PKA phosphorylated GST-C/EBPϵ. (C) p38 MAP kinase phosphorylated GST-C/EBPϵ. (D) p38 MAP kinase phosphorylated GST-C/EBPϵT75A. (E) A diagrammatic representation of cold phospho–amino acid standards on ninhydrinstained TLC plate. Arrows indicate the location of phosphorylated amino acid residues.