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. 2004 Mar;72(3):1291–1297. doi: 10.1128/IAI.72.3.1291-1297.2004

FIG. 4.

FIG. 4.

B. anthracis inhibits IFN-α/β signaling. Human macrophages were incubated with the Sterne strain (34F2) with either IFN-β at 1,000 U/ml, IFN-γ at 400 U/ml, or saline at an MOI of 20:1 for 4.5 h. (A) Immunoblot for ptyr701-STAT1 and total STAT1. Infection of human AM with 34F2 failed to induce ptyr701-STAT1 at 4.5 h (compare lanes 1 and 4) and reduced the degree of STAT1 phosphorylation by IFN-β twofold compared to uninfected IFN-treated controls (compare lanes 3 and 6). 34F2 had a minimal effect on IFN-γ-induced ptyr701-STAT1 formation (compare lanes 2 and 5). (B) Immunoblot for pser727-STAT1 and total STAT1 from STAT1-immunoprecipitated cell lysates. Infection of human THP-1 macrophages with 34F2 failed to induce pser727-STAT1 at 4.5 h (compare lanes 1 and 4) and reduced the degree of STAT1 phosphorylation by IFN-β twofold compared to uninfected IFN-treated controls (compare lanes 3 and 6). 34F2 had a minimal effect on IFN-γ-induced pser727-STAT1 formation (compare lanes 2 and 5). (C) EMSA for ISGF-3. Infection of human AM with 34F2 failed to induce ISGF-3 DNA binding at 4.5 h (compare lanes 1 and 3) and inhibited IFN-β-induced ISGF-3 production (compare lanes 2 and 4). This band was confirmed to be ISGF-3 in a separate experiment by both cold competition (lane 6) and supershift with antibodies to STAT1, STAT2, and IRF-9 (lanes 7 to 9). (D) EMSA for STAT1 homodimer (GAS binding). IFN-γ induced STAT1 homodimer formation (lane 2), which was unaffected by infection of human AM with 34F2 (lane 5). This complex was supershifted with STAT1 antibody (lane 3). In all cases lanes were normalized for total protein.