Nef-dependent activated STAT1 forms homodimers and binds specific DNA-responsive elements.
(A) EMSA performed by using total cell extracts from MDMs 8 hours after the infection with either Δenv or Δenv/nef(VSV-G) pseudotyped HIV-1 (A,B) or after treatment with 100 ng/mL rNef for the indicated times (C,D), and the 32P-labeled GAS element of the human IRF-1 promoter (A-C), or the 32P- labeled SIE element of the human c-fos promoter (B-D) as a probe. As controls, GAS (A-C) or SIE competitors (B-D) were used. Supershifts were performed with anti-STAT1 antibodies, and also with anti-STAT3 antibodies (B-D). As an additional control, SIE-specific EMSA was also performed on total extracts of HeLa cells treated with 100 IU/mL IFN-β (D). Electrophoretic mobility is indicated on the left side for STAT1-1/GAS complexes and on the right side for STAT1-1, 1-3, and 3-3/SIE complexes.
