FIG 3.

Inhibition of transcription is permissive for Y701 dephosphorylation of nucleoplasmic STAT1. (A) STAT1 and STAT2 tyrosine dephosphorylation in the absence of Irf9. WT and Irf9^−/− BMDMs were stimulated with IFN-β for the indicated times. Levels of tyrosine-phosphorylated STAT1 and STAT2, total STAT1 and STAT2, and tubulin (used as a loading control) were assessed by Western blotting. Note that total STAT1 and STAT2 levels strongly increased with time of IFN-β treatment in Irf9^−/− cells, in contrast to levels in WT cells (compare the 0-min with the 360-min time points). (B) IFN-β-activated STAT1 accumulates in the nucleus in the absence of IRF9. Irf9^−/− BMDMs were treated with IFN-β for the indicated times, and immunofluorescence analysis was performed using STAT1-specific antibodies. (C) IFN-β-induced transcription of Irf1 proceeds in the absence of Irf9. WT and Irf9^−/− BMDMs were treated with IFN-β, and total RNA was isolated and was analyzed by qRT-PCR. Error bars indicate standard deviations for 3 biological replicates. (D) Ongoing tyrosine dephosphorylation of STAT1 and STAT2 in Irf9^−/− BMDMs with a block in protein synthesis. Irf9^−/− BMDMs were treated as described in the legend to panel A in the presence or absence of CHX and were analyzed by Western blotting. (E) Amounts of STAT1 and STAT2 heterodimers remain stable regardless of IFN-β treatment. BMDMs were stimulated with IFN-β for the indicated times, and immunoprecipitation (IP) was carried out using antibodies against STAT1 (top) or STAT2 (bottom), or beads only as a control (CTRL). Immunoprecipitated complexes were analyzed by Western blotting using antibodies to STAT1 and STAT2. (IN, input control [20%]). (F) STAT1 and STAT2 form complexes in Irf9-deficient cells. STAT1 was immunoprecipitated in Irf9^−/− BMDMs as described in the legend to panel E. (G) Y701 dephosphorylation of WT and DNA binding-deficient STAT1 after IFN-γ stimulation. Immortalized MEFs expressing either WT STAT1 or a DNA binding-deficient STAT1 mutant (K336A) were stimulated with IFN-γ for the indicated times and were analyzed by Western blotting as described for panel A. (H) Y701 dephosphorylation of the DNA binding-deficient STAT1 mutant. MEFs expressing STAT1 K336A were stimulated with IFN-γ for 5 min (“pulse”), followed by a chase for the indicated times in the presence or absence of the JAK2 inhibitor P6. (I) Y701 dephosphorylation of MEFs expressing WT or K336A STAT1 after stimulation with IFN-β or IFN-γ. MEFs expressing WT or K336A STAT1 were pulsed with IFN-β or IFN-γ for 5 min, followed by a chase for the indicated times. STAT1 tyrosine phosphorylation was analyzed by Western blotting as described for panel A.