Extended Data Fig. 1: IFN-α-dependent proximity of IFNAR1 and Hrs on endosomes.
a, Immunofluorescent labelling of Hrs, EEA1 and endocytosed IFNAR1 subunits after 10 min IFN-α (upper panels) or IFN-β (lower panels) stimulation in eGFP–Rab5 Q79L-transfected RPE1 cells observed by confocal microscopy with representative plots of co-localization profiles on endosomes. Quantification of Hrs and IFNAR1 co-localization is expressed as the Manders’ coefficient indicating the portion of IFNAR1 pixels containing Hrs pixels. Scale bar, 10 µm. Data represent the mean ± s.e.m., n = 8 cells examined from two independent experiments. Statistical significance was determined using a two-tailed Student’s t-test. ***P < 0,001; NS, not significant. b, Fast FLIM analysis of the IFNAR1–eGFP fluorescence lifetime in segmented endosomes of transfected RPE1 cells (with or without Hrs–mCherry expression) at steady state or following stimulation with IFN-α or IFN-β for 10 or 20 min (one dot is the mean lifetime of all endosomes from one cell); n = 28, 29, 29, 29, 29 and 29 cells, respectively, examined from four independent experiments. Data are the median ± 95% confidence interval. P values were calculated using a one-way ANOVA with the Kruskal–Wallis multiple comparison test. ***P < 0.001; NS, not significant. Source numerical data are provided.
