FIGURE 6.

Analysis of MK2 activation/activity in P. falciparum GPI-stimulated macrophages. WT macrophages were plated in 24-well plates (∼5 × 10⁵ cells/well). After overnight culturing, the cells were treated with U0126, SB203580, and U0126 plus SB203580 or PD98059 as indicated for 1 h and stimulated with 200 nm GPIs (A, lanes 2–11), 10 ng/ml Pam₃CSK₄ (B, lanes 2–11), or 100 ng/ml LPS (data not shown) for 1 h. Lane 1, unstimulated control cells. The cells were lysed with 30 μl of RIPA buffer and lysates electrophoresed on 8% SDS-polyacrylamide gels. The proteins bands on gels were transferred onto nitrocellulose membranes, and membranes were treated with anti-peptide antibodies specific to phosphorylated (P) and nonphosphorylated forms of MK2. The nonphosphorylated MK2 bands served as loading control. The bound antibodies were detected with HRP-conjugated secondary antibodies (1:2000 dilution) and chemiluminescent substrate system. C, WT macrophages (∼5 × 10⁶/flask) were treated with U0126, SB203580, or U0126 plus 10 μm SB203580 for 1 h. Untreated cells were used as controls. The cells were stimulated with 100 nm GPIs for another 1 h, lysed with 500 μl of lysis buffer, and aliquots of lysates were analyzed for MK2 kinase activity with HSP27 as the substrate using MK2 immunoprecipitation kinase assay kit according to the manufacturer's procedure. Phosphorylated HSP27 was analyzed by Western blotting using HSP-Ser-78 phospho-specific antibody (1:1000 dilution).