Fig. 6

Localization of STAT complexes in BMDM. a IRF9 localization as determined by immunofluorescence. BMDMs of wild-type (WT) and Irf9^−/− (IRF9^−/−) mice were left untreated or stimulated with IFN-β for 30 min. The cells were fixed and stained with an anti-IRF9 antibody followed by Alexa Fluor^® 488-conjugated secondary antibody (green). Nuclei were stained with DAPI (magenta). First Ab (−) indicates the control without the first antibody. The scale bars represent 10 µm. b–d Nuclear and cytoplasmic extracts from BMDM were prepared from controls or after a 30-min treatment with IFN-β or IFN-γ and analyzed by western blot. A 2:1 ratio of the nuclear-to-cytoplasmic fraction is shown. Where indicated, 15 µM P6 inhibitor or DMSO were added for 3 h prior to IFN treatment. Phosphorylation of STAT1 at Y701 and total STAT1 levels, as well as phosphorylation of STAT2 at Y689, total STAT2 and IRF9, and α-tubulin and lamin A/C levels were determined. e The nuclear fractions of the representative blots b–d were quantified using ImageJ software. Relative intensities of the bands were normalized to their corresponding lamin C levels. Data represent relative intensities in percent, where STAT1, STAT2, and IRF9 levels in IFN-β-treated nuclear extracts equal 100%. f Phosphorylation of STAT1 at Y701, STAT2 at Y689, total STAT1, total STAT2, IRF9, and α-tubulin was determined in whole-cell lysates of BMDM. Overall, 15 µM P6 inhibitor or DMSO were added for 3 h prior to IFN treatment. Source data are provided as a source data file