FIGURE 7.

Induction and nuclear translocation of transcription factors in P. falciparum GPI-stimulated macrophages. A, WT and MK2^−/− macrophages were plated in T-25 flasks (5–10 × 10⁶ cells/flask). After overnight culturing, the cells were stimulated with 100 nm P. falciparum GPIs. At the indicated time points, the cells were harvested, and nuclear extracts (50 μl total volume) were prepared as described under “Experimental Procedures.” The extracts (20 μl) were electrophoresed on 10% SDS-polyacrylamide gels, and protein bands were transferred onto nitrocellulose membranes and treated with 1 μg/ml anti-IκBζ, anti-p50, anti-p65, anti-c-Rel, or anti-β-actin antibodies. The bound antibodies were detected with HRP-conjugated secondary antibodies (1:2000 dilution) and chemiluminescent substrate system. B, WT macrophages (5–10 × 10⁶ cells/flasks) were treated with 10 μm SB203580 for 1 h. Untreated cells were used as controls. The cells were stimulated with 100 nm P. falciparum GPIs for 3 h and harvested, and nuclear extracts were collected. Aliquots of lysates were analyzed by Western blotting as outlined in A. The experiments were repeated three times, and the results of representative experiments are shown.