IL-3 or forskolin stimulated phosphorylation of CREB occurs independently of p38 MAPK and MAPKAP kinase-2. (A) MC/9 cells were treated with SB203580 (1 μmol/L) or vehicle alone for 20 minutes, followed by treatments with either 10 μg/mL synthetic IL-3, 50 μmol/L C2-ceramide, or both, for 5 minutes. Whole-cell lysates were separated on SDS-PAGE and immunoblot analysis was performed to detect phospho-CREB. (B) MC/9 cells were treated with 10 μg/mL synthetic IL-3 for the times indicated following pretreatment with SB203580 or vehicle alone for 20 minutes. Immunoblot analysis was performed to detect phospho-CREB (top) and CREB (bottom). (C) MC/9 cells were treated with 10 μg/mL IL-3 for the times indicated, following treatment with SB203580 (1 μmol/L) or vehicle alone for 20 minutes. Lysates were incubated with 1 μg anti-MAPKAP kinase-2 antibody and protein G-Sepharose beads, and kinase activity was determined from the washed immunoprecipitates by measuring the³²P transferred to Hsp25. (D) Cells were treated with the indicated concentrations of forskolin for 10 minutes following a 20-minute treatment with SB203580 (1 μmol/L) or vehicle alone. Immunoblot analysis was performed to detect phospho-CREB (top) and CREB (bottom).