Functional impact of the variant on non-canonical and canonical NF-κB pathways. (A) Dilution histogram of Cell Trace Violet dye (CTV) after stimulation of activated T cells with OKT3 at 1 or 2 µg/ml for 4 days or anti-CD3/CD28 beads for 4 days. Representative of two independent experiments. (B) Histogram of CD69 activation marker upregulation on activated T cells after 4 days of stimulation with either OKT3 or anti-CD3/CD28 beads. Patient is in red, and controls are in gray. Representative of two independent experiments. (C) Western blot of cytoplasmic and nuclear extracts from SV40-fibroblasts after 48 h of LTαβ (LT) or TNFα (TNF) stimulation. RELB was revealed using a specific antibody to assess RELB production and nuclear translocation. α-Tubulin was used the loading control of cytoplasmic extract while Lamin A/C was used for nuclear extracts. The patient is indicated in red, and the controls are C1, C2, and C3. Representative of two independent experiments. (D) Electromobility shift assay on nuclear extracts from healthy control and patient SV40-fibroblasts after different time points of LTα1β2 stimulation showing the absence of RELB binding (red arrows) in patient as compared to controls (lower thin band) and the normal binding of RELA (upper band). Representative of three independent experiments. (E) Relative VCAM1 mRNA expression after 24 h of LTαβ relative to non-stimulated condition. These experiments have been performed on healthy controls and patient SV40-fibroblasts. *: P-value <0.05 after Mann-Whitney-Wilcoxon test. All figures are representative of at least two independent experiments. Source data are available for this figure: SourceData FS3.