Figure 1

Isolation of C/EBPβ phosphorylated by p38α or p38β MAPK for tryptic phosphopeptide mapping. HEK293T cells were co-transfected with plasmids encoding C/EBPβ (LAP fused with the FLAG tag) and constitutively active p38α or p38β (fused with the HA tag). After 48 h incubation the cells were lysed. Expression of the transfected plasmids was verified by western blot analysis of HA and FLAG. Activity of over-expressed p38α and p38β was evaluated by western blot analysis of ATF2 activation (A). Overexpressed C/EBPβ was pulled down with FLAG-M2 magnetic beads and separated by SDS-PAGE. The gel was stained with Coommassie Blue R-250 (B). The C/EBPβ band was then cut out and analyzed by tryptic phosphopeptide mapping utilizing mass spectrometry for phosphorylated amino acid residues. MAPK, mitogen-activated protein kinase.