FIG. 5.
IFN-γ induces a sustained STAT1 activation only in mouse but not human fibroblasts, and VACV incompletely interferes with STAT1 activation in mouse fibroblasts. EMSA analysis using a 32P-labeled GAS probe reveals a long-lasting formation of STAT1 homodimers in IFN-γ-pretreated mouse NIH 3T3 (right panels) but not human MRC-5 (left panels) fibroblasts. Cells were mock infected or infected at 5 PFU/cell with VACV for 6 h. Cells were incubated with 500 U/ml IFN-γ for 30 min or preincubated with 500 U/ml IFN-γ for 48 h. Lysates were fractionated and equivalent amounts of nucleoplasmic or cytoplasmic extracts were subjected to EMSA analysis. STAT1 protein complexes were identified by shifting when adding a STAT1-specific antibody. STAT1 complexes are indicated by arrowheads. The detection of proteins by immunoblotting was performed with the same lysates. Antibodies specific for human and P-Tyr STAT1 were used. Immunoblots were reprobed for STAT1 and β-actin.