C2-ceramide stimulates p38 MAPK activity . MC/9 cells were treated with either synthetic IL-3 (10 μg/mL) for 5 minutes, C2-dihydroceramide (DH; 50 μmol/L for 5 minutes, or C2-ceramide (50 μmol/L) for the indicated times. Cells were then detergent-solubilized and p38 MAPK was immunoprecipitated from the lysates with -p38 MAPK antibody bound to protein A-Sepharose beads. (A) Activity of the washed immunoprecipitates was determined by³²P incorporation into a peptide corresponding to amino acids 1-96 of ATF-2. Immunoblot analysis was performed to detect phosphotyrosine (4G10; B) and with -p38 MAPK (C) to confirm equal amounts of p38 MAPK in the immunoprecipitates.