Macrophage-Activating Lipopeptide-2 Requires Mal and PI3K for Efficient Induction of Heme Oxygenase-1
Figure 3
MALP-2 induces HO-1 expression in a Btk-dependent manner.
A. THP-1 cells were stimulated with MALP-2 for the indicated time intervals, the cell lysates were subjected to Western blotting using an anti-phospho-Btk at Tyr223. *, P<0.05 and **, P<0.01 as compared with the basal level. B. Cells were pretreated with or without PP1 for 1 h, and then incubated in the absence or presence of 5 ng/ml MALP-2 for 10 min. Cell fractions were prepared and subjected to Western blotting analysis with an anti- phospho-Btk antibody. Data shown represents three experiments (means ± SEM). C. Cells were transfected with siRNA of c-Src, and then treated with MALP-2 for 10 min. Phosphorylated Btk was detected by Western blotting. Results shown are representative of at least three separate experiments. D. THP-1 cells were pretreated with a Btk inhibitor FLM-A13 (30 µM and 100 µM) for 1 h, and incubation was continued with MALP-2 (5 ng/ml) for another 16 h. Samples were subjected to Western blotting for the detection of HO-1 expression. Densitometry analysis was performed on at least three Western blots quantified by scanning densitometry. E. Cells were transiently transfected with HO-1-luc reporter gene, and then pretreated with LFM-A13. After incubation for 1 h, cells were stimulated with MALP-2 (5 ng/ml) for 8 h. The luciferase activity derived from HO-1 activation was normalized to the transfection efficiency with β-gal. Data represent means ± SEM from at least three independent experiments. *, P<0.05 and **, P<0.01 for significant difference between compared groups.