Activation of p38^SAPK in purified neutrophils.

(A) PMN were stimulated with 1 μM FMLP as indicated in the figure, and p38^SAPK was immunoprecipitated and subjected to a kinase assay using glutathione S-transferase–tagged MAPKAP kinase-2 as substrate in the presence of γ–³²P-ATP. Samples were then subjected to Western blotting and autoradiography as described in “Materials and methods.” The top panel shows the autoradiograph of the region of the blot corresponding to MAPKAP kinase-2. The bottom panel shows the membrane reprobed with a p38^SAPK-specific antibody to check for equal loading of p38 kinase in each lane. PMN were also stimulated with (B) TNF-α and (C) GM-CSF followed by preparation of cell lysates, separation of proteins by SDS-PAGE, and immunoblotting with antibodies specific for phosphorylated p38^SAPK (top panel) and total cell p38^SAPK(bottom panel). (D) Neutrophils (2 × 10⁷ cells per mL) were incubated with the p38 kinase inhibitor SB203580 at various doses or DMSO diluent prior to stimulation with FMLP for 1 minute. Preparation of p38^SAPK immunoprecipitates and p38 kinase assay was performed as described in panel A. The top panel shows the autoradiograph of the region of the membrane corresponding to MAPKAP kinase-2, and the bottom panel shows the membrane reprobed with a p38^SAPK-specific antibody.