Reconstitution of patient cells with IKKα restored the non-canonical NF-κB defect in vitro. (A and B) Patient cells were transduced with an EV (P+EV) lentivirus or containing the coding sequence of wt IKKα (P+wt). GFP-positive cells were sorted, and cell lysate after activation of the non-canonical pathway by LTαβ (A) or Tweak (B) were subjected to western blot. p100 and p52 were revealed using specific antibodies and GAPDH was used as a loading control. Representative of three independent experiments. (C) Patient cells were transduced with an EV (P+EV) lentivirus or containing the coding sequence of wt IKKα (P+wt). GFP-positive cells were sorted. After activation with either LTαβ or Tweak, cytoplasmic and nuclear protein separation was performed and subjected to western blot. RELB was assessed using a specific C-terminal antibody to assess its nuclear translocation after activation in transduced patient’s cells compared with controls. α-Tubulin was used as a loading control of cytoplasmic extract while Lamin A/C was used for nuclear extracts. RELB protein expression was quantified and normalized to the quantity of Lamin A/C in arbitrary units. Representative of three independent experiments. Source data are available for this figure: SourceData F6.