Biochemical characterization of S. mansoni p38 MAPK. (A) Immunodetection of phosphorylated S. mansoni p38 MAPK. Protein from astrocytoma (U251 MG) cells (lane 1), 900 freshly-hatched swimming miracidia (lane 2), or an adult worm pair (lane 3) was processed for western blotting using anti-phospho p38 MAPK antibodies. (B) Activated S. mansoni p38 MAPK phosphorylates activating transcription factor-2 (ATF-2), and SB 203580 inhibits p38 MAPK activity. p38 MAPK from adult worm pairs was immunoprecipitated using immobilized anti-phospho p38 MAPK antibodies and the immunoprecipitated protein used in an in vitro kinase assay to phosphorylate ATF-2 either in the presence of SB 203580 (1-5 μM) or DMSO (D+), or with neither (D-). Phosphorylation of ATF-2 by immunoprecipitated p38 MAPK was assessed by western blotting using anti-phospho ATF-2 antibodies. (C) Anisomycin activates p38 MAPK in S. mansoni miracidia. Freshly-hatched miracidia were exposed to anisomycin (20 μM) for various durations and phosphorylation of p38 MAPK in miracidia detected by western blotting with anti-phospho p38 MAPK antibodies; blots were also probed with anti-actin antibodies to demonstrate equal protein loading between lanes. Results shown in (A-C) represent those obtained in at least two independent experiments.