Extended Data Fig. 5. The processing-resistant NFKB2 mutants have enhanced p100-IκBδ activity in heterozygous patients’ cells.

(a) Western blot of P-p100, NF-κB2 (p100/p52), NF-κB1 (p105/p50), RelB, and RelA in primary fibroblasts from one healthy donor (HC), a patient with the p52^LOF/IκBδ^GOF R853*/WT variant, a patient with the p52^LOF/IκBδ^LOF K321Sfs/WT variant, a patient with AR complete (Q72Tfs*152/Q72Tfs*152) RelB (RelB^−/−) or (P565R/P565R) NIK (NIK^−/−) deficiency, with or without stimulation with LT-α1β2 (Lt) for 48 h (left panel), and a graph depicting total p100/p52 intensity ratio after Lt stimulation (right panel). Bars represent the mean values (± s.d.) from two independent experiments. (b) Western blot showing p100 processing into p52 and RelB induction in total cell extracts of SV40 fibroblasts from a healthy donor (HC) or a patient with AR complete NIK deficiency (NIK^−/−) either left non stimulated (NS) or after stimulation for 48 h with TNF, TWEAK, Lt. Data representative of three independent experiments are shown. (c) Confocal microscopy showing the subcellular distribution of RelB in primary fibroblasts from two healthy controls (HC1, HC2), patients with a p52^LOF/IκBδ^LOF K321Sfs/WT or a p52^LOF/IκBδ^GOF R853*/WT NF-κB2/p100 variant, and patients with AR complete RelB (RelB^−/−) or NIK (NIK^−/−) deficiency, without and with stimulation with TWEAK for 48 h. Data representative of three independent experiments are shown. (d) Confocal microscopy showing the subcellular distribution of p100/p52 in primary fibroblasts from two healthy controls (HC1, HC2), patients with a p52^LOF/IκBδ^LOF K321Sfs*/WT or a p52^LOF/IκBδ^GOF R853*/WT NF-κB2/p100 variant, AR complete RelB deficiency (RelB^−/−), or AR complete NIK deficiency (NIK^−/−) without and with stimulation with TWEAK for 48 h, with an antibody recognizing the N-terminus of p100. Data representative of three independent experiments are shown. The bottom panels represent magnified images (cropped images).