Acetylation affects IFNα-induced phosphorylation and translocation of STAT1. (A) STAT1 was immunoprecipitated from lysates of 293T cells treated with IFNα for the time periods indicated. Acetylation, phosphorylation, and precipitation of STAT1 were analyzed by Western blot; (IP) immunoprecipitation; (pre) preimmune serum IP. STAT1 phosphorylation was analyzed with an antibody specifically recognizing STAT1 phosphorylated at Y701 in this and all following experiments when indicated. (B) 293T cells were incubated with IFN-α for 40 min (+). Anti-acetyllysine IPs formed under stringent conditions with RIPA buffer (1% SDS) were analyzed for the presence of pY701-STAT1 by Western blot (IP with control IgG). (C) 293T cells were incubated with IFNα for 20 min or remained untreated (0). One-hundred-eighty minutes later, cells were restimulated for 20 min (180 + 20 min). Parallel cultures were kept naive and treated with IFNα for only 20 min. Cell lysates were probed for STAT1 phosphorylation and expression in Western blot. (D) As in C, except that the protein/RNA synthesis inhibitors Cycloheximide (CHX) and Actinomycin D (ActD), or the proteasomal inhibitor MG-132, had been added 1 h before initial IFN treatment. (E) 293T cells were incubated with HDACi [(V) VPA; (T) TSA] or left untreated (C). Twenty-four hours later, cells were stimulated with IFNα for 20 min (+). Lysates were analyzed as in C. (F) 293T cells were coincubated with IFNα and HDACi for up to 3 h. Lysates were analyzed as in C. (G) U3A cells were transfected with DNA for STAT1 (WT), lysine mutants K410,413Q (QQ), K410,413R (RR) , or empty vector pcDNA3.1 (3.1); (2f) 2fTGH, STAT1-positive U3A parental cell line. Forty-eight hours later, IFNα was added to the cells for 1 h (+). The Western blot was probed as indicated. (H) Translocation of EGFP-STAT1 (WT) and indicated K410,413 mutants was assessed by live cell time-lapse fluorescence microscopy. Transfected U3A cells were treated with IFNα for 0–60 min. In this and all following microscopy experiments, the bar corresponds to 10 μm if not stated otherwise. (I) Binding of STAT1 (WT/QQ/RR) to Importin α5 was analyzed by GST pull-down with lysates of transfected U3A cells. Western blots were probed for STAT1 and GST-Importin α5. Cells were treated for 1 h. Pull-down with GST alone did not precipitate STAT1 (not shown). (J) Confocal immunofluorescence microscopy shows colocalization of transfected wild-type (WT) and pseudo-acetylated STAT1 (QQ) with endogenous TYK2, JAK2, or IFNAR1/2 in U3A cells (numbers state “overlap equation” r0). (K) Wild-type STAT1 and K410,413Q were equally recovered in IFNAR2 IPs formed from lysates of transfected U3A cells. (L) U3A cells were transfected with plasmids for STAT1 (WT) or STAT1K410,413R (RR). The time course of STAT1 phosphorylation and expression, and IFN-induced nuclear translocation of EGFP-tagged STAT1 (WT/RR) was analyzed 180 min after IFN stimulation. (M) U3A cells were transfected with DNA for STAT1 (WT) or STAT1K410,413R (RR). Forty-eight hours later, IFNα was added for 3 h (+). Lysates were prepared under conditions disrupting protein–protein interactions (1% SDS). IPs formed with anti-STAT1 or anti-acetyllysine antibodies were probed for acetyllysine and STAT1, or STAT1, respectively. (N) GST pull-downs with the indicated fragments of CBP on lysates of U3A cells reconstituted with STAT1 or STAT1K410,413R by Western blot against STAT1 or GST.