FIG 7.

Processive transcription is required for downregulation of STAT1 promoter occupancy but not for Y701 dephosphorylation. (A) Pervanadate (Na₃VO₄) inhibits the tyrosine dephosphorylation of STAT1 and STAT2. BMDMs were stimulated with IFN-β in the presence or absence of Na₃VO₄ for the indicated times. Cell extracts were analyzed by Western blotting using antibodies to Y701-phosphorylated STAT1 (pY-STAT1) and STAT2 (pY-STAT2), total STAT1 and STAT2, and tubulin (used as a loading control). (B) Na₃VO₄ treatment results in persistent STAT1 nuclear accumulation. BMDMs were stimulated with IFN-β in the presence or absence of Na₃VO₄ for the indicated times. Cellular localization of STAT1 was assessed by immunofluorescence using STAT1-specific antibodies. (C and D) Inhibition of tyrosine dephosphorylation does not prevent a decrease in STAT1 promoter occupancy. BMDMs were stimulated with IFN-β in the presence or absence of Na₃VO₄ for the indicated times. STAT1 recruitment was assessed by ChIP for Irf1-GAS (C) and Mx2-ISRE (D) sites. Signals were normalized to input DNA. Error bars represent standard deviations (n = 3). (E) Na₃VO₄ treatment is permissive for IFN-β-induced transcription. BMDMs were stimulated with IFN-β in the presence or absence of Na₃VO₄. Total RNA was isolated, and the levels of Irf1, Mx2, Socs1, and Ifit1 mRNAs were quantified using qRT-PCR. Error bars indicate standard deviations (n = 3).