FIGURE 9.

Analysis of c-Maf expression and GAP-12 binding to IL-12p40 gene promoter in P. falciparum GPI-stimulated macrophages. A, WT and MK2^−/− macrophages (5–10 × 10⁶ cells/flask) were stimulated with 200 nm GPIs for 1, 2.5, and 4 h and harvested; nuclear extracts (50 μl total volume) were prepared, and 20-μl aliquots were analyzed by Western blotting using anti-c-Maf antibody (1 μg/ml). The nuclear extracts were also analyzed for β-actin expression as loading control using anti-β-actin antibody. B, WT and MK2^−/− macrophages were stimulated with 100 nm GPIs for 2.5 and 4 h, and nuclear extracts were prepared as outlined in Fig. 8. Aliquots of nuclear extracts (2 μl) were incubated with biotin-labeled GAP-12 DNA probe in binding buffer (20 μl). The reaction mixtures were electrophoresed on 6% polyacrylamide gel and transferred onto nylon membranes, and GAP-12 protein bound to DNA probe was detected using HRP-conjugated streptavidin and chemiluminescent detection reagent. The numbers on the top of electrophoretograms indicate the relative band intensities of nuclear C-Maf (A) and GAP-12-DNA complex (B) determined by scanning of x-ray films using Bio-Rad GS-800 densitometer.