Fig. 3.

Thr₁₈₈ of C/EBPβ is phosphorylated both by MAPK and cdk2/cyclinA. (A) rC/EBPβ was incubated without or with MAPK or cdk2/cyclinA and [γ-³²P]ATP. After SDS/PAGE, [³²P]C/EBPβ was detected by autoradiography (Upper), and total C/EBPβ was detected by Coomassie blue staining (Lower); Similar experiments were performed as above but with cold ATP. (B) Immunoblotting was with anti-P-Thr₁₈₈-C/EBPβ (Upper) or anti-C/EBPβ (Lower) antibodies. (C) The C/EBPβ band was excised from the gel, digested by trypsin, and analyzed by nanoLC-MS/MS. A singly phosphorylated peptide species from C/EBPβ was observed (m/z 1,094.8) with MAPK or cdk2/cyclinA (Middle and Bottom, respectively). No phosphorylation was observed in the same peptide (m/z 1,067.5) without kinase treatment (Top).