Figure 7. ESAT-6 stimulates STAT3 activation and IL-6 production by human macrophages.

Human monocyte derived macrophages were incubated with 1 μg/ml ESAT-6 for different periods and phospho-STAT3 and phospho-STAT1 were determined by Western blotting followed by blotting for total STAT3 and STAT1 after stripping (A) in cellular protein extracts. The DNA binding activity in the cellular protein extracts was determined by EMSA using labeled STAT3 as probe. The specificity of binding and identity of STAT3 was confirmed by competition and supershift EMSA as indicated (B). The arrow indicates specific DNA-protein complex. Some cells were stimulated with ESAT-6 at indicated concentrations and IL-6 levels were determined in 24 h culture supernatants (C). Human macrophages were infected with different strains of Mtb as described in Fig. 6A and IL-6 levels were determined at 24 and 48 h later (D).