C/EBPϵ is phosphorylated in vitro by ERK2, PKA, and p38 MAP kinase. GST-C/EBPϵ was in vitro kinased by the individual protein kinases and the reaction separated on SDS-PAGE gel and transferred to PVDF membrane. The following enzymes were used: (A) ERK2; (B) p38α MAP kinase; (C) PKA; (D) JNK2; (E) casein kinase 2; (F) calcium-calmodulin kinase II. (i) Phosphorylation was visualized by autoradiography. (ii) The same membrane was subsequently probed with an antibody specific for C/EBPϵ and the results were visualized by enhanced chemiluminescence (ECL; Amersham).