p.R25S underlies IRF4 deficiency. (A) Upper panel: Western blot for IRF4, lamin A/C, and vinculin on cytoplasmic (C) and nuclear (N) extracts from HEK293T cells not transfected (NT) or transfected with EV or various IRF4 cDNAs. Lower panel: Quantification of the IRF4 nuclear/cytoplasmic expression ratio based on band intensity analysis from western blots. Data representative of two independent experiments are shown. (B) EMSA and supershift assays with the nuclear extract of HEK293T cells transfected with EV or various IRF4 cDNAs and incubated with a fluorescent ISRE probe. The IRF4–ISRE complex and the supershifted IRF4–ISRE complex are indicated with arrows. Representative data from two independent experiments are shown. (C) Dual luciferase ISRE-IFNB1 reporter activity of HEK293T cells transfected with EV or with various IRF4 cDNAs. Data from eight independent experiments performed in triplicate are shown. Dotted-line represent the normalization to EV. (D) Upper panel: Western blot for IRF4, lamin A/C, and vinculin on cytoplasmic (C) and nuclear (N) extracts of EBV-B cells from three healthy controls (CTLs), patients, and an individual heterozygous for the p.R98W IRF4 variant. Lower panel: Quantification of the IRF4 nuclear/cytoplasmic expression ratio based on band intensity analysis from western blots. Representative data from three independent experiments are shown. Source data are available for this figure: SourceData F2.