Expression of IFNAR1 variants and their impact on the response to type I IFNs. (A) Western blotting for IFNAR1 in IFNAR1-deficient HEK293T cells transiently transfected with WT or mutant IFNAR1 cDNA constructs. An antibody recognizing the N-terminus of the IFNAR1 protein was used. GAPDH was used as a loading control. A representative blot from at least two experiments is shown. NT, non-transfected; EV, empty vector. (B) Flow cytometry histogram of cell-surface IFNAR1 levels in IFNAR1-deficient HEK293T cells transiently transfected with WT or mutant IFNAR1 cDNA constructs and then subjected to extracellular staining with a specific antibody recognizing the N-terminal part (SD2) of the IFNAR1 protein. All histogram plots are representative of at least two independent experiments. (C) IFNAR1-deficient HEK293T cells were transiently transfected with WT or mutant IFNAR1 cDNA constructs and were then stimulated with the indicated IFNs for 24 h, and luciferase activity was measured. The IFN-α subtypes are arranged in order of affinity for IFNAR1 binding, from the highest (left, IFN-α8) to the lowest (right, IFN-α17) affinity (Table S1). The heatmap shows the mean luciferase activity relative to the WT from two independent experiments. Source data are available for this figure: SourceData F3.