Impact of the NEMO D311G substitution on the CD40-dependent NF-κB pathway. (A) IκBα degradation was observed by Western blot analysis on cytoplasmic protein extracts. Antibodies against GAPDH were used for normalization of protein levels on Western blots, and (B) NF-κB translocation was observed by EMSA on nuclear protein extracts from EBV-B cells activated with CD40L (100 ng/mL) for various times from a healthy control subject (C⁺), a patient with complete CD40 deficiency (CD40^−/−), a hypomorphic NEMO EDA-ID-OL patient (X420W), an NEMO EDA-ID patient with another mutation affecting position 311 (D311N), the proband (P [D311G]), and an NEMO XR-MSMD patient (R319Q). These results are representative of 2 independent experiments. (C) The production of IL-12p40, as assessed by ELISA, in MDDCs activated or not (NS) for 24 hours with trimeric soluble CD40L (1 μg/mL) from healthy control subjects (C⁺; n = 8), a patient with complete CD40 deficiency (CD40^−/−), an NEMO EDA-ID patient with another mutation in position 311 (D311N), the proband (P [D311G]), and an NEMO XR-MSMD patient (R319Q). NS indicates not stimulated.