Fig. 4.

Signal dependence of complex formation from ISGF3 subunits. a BMDMs from wild-type (WT) animals were treated for 1.5 h with IFN-β and immunoprecipitation was carried out using antibodies against STAT1, STAT2, IRF9, or an IgG control. Immunoprecipitated complexes were analyzed by western blotting with antibodies to STAT1, STAT2, IRF9, and GAPDH. Input controls represent 10% of the total lysate used for the immunoprecipitation. b The representative blot was quantified using ImageJ software. Relative intensities of the bands were normalized to their corresponding input sample. Data represent relative intensities in percent, where STAT1, STAT2, and IRF9 levels in IFN-β-treated IPs equal 100%. c Targeted quantitative MS analysis of STAT1, STAT2, and IRF9 BioIDs using PRM. Raw 264.7 cells were treated with 0.2 µg/ml doxycycline for 24 h, followed by addition of 50 µM biotin for 18 h. Cells were treated for 2 h either with IFN-β or IFN-γ lysed and protein complexes were isolated by streptavidin affinity purification, followed by analysis with LC–MS. Mean log₂-transformed protein ratios were calculated for three biological replicates of myc-STAT1-BirA*, myc-STAT2-BirA*, or myc-IRF9-BirA* cells normalized to their appropriate localization control. Standard deviation and t-test statistics were calculated for each of the target proteins. P-values (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). Source data are provided as a source data file