Contribution of IKKβ(R286X) mutation to CID. (A-C) Patients’ blood lymphocytes responded normally when stimulated with phytohemagglutinin (PHA) but had variably low responses to concanavalin A (Con A) and pokeweed mitogen (A). However, they failed to respond when stimulated with candida and tetanus antigens (B) or soluble or immobilized anti-CD3 (C). (D) Impaired upregulation of T-cell activation markers in patients’ CD4 T cells after overnight stimulation with plate-bound anti-CD3 or phytohemagglutinin. (E-F) Neither full-length nor truncated mutant IKKβ(R286X) protein is detectable in patients (PT), siblings, and normal peripheral blood mononuclear cells (E) and EBV-transformed B cells (F) by immunoblotting analysis with anti-N- and anti-C-terminal IKKβ antibodies. (G) mRNA levels of IKKα, IKKβ, and NEMO in PMCs detected by real-time quantitative polymerase chain reaction. (H) IKKβ(R286X) is not able to form a complex with IKKα/NEMO. Cell lysates from Phoenix-Eco cells transfected with Flag-tagged-FL-IKKβ, Flag-tagged-IKKβ(R286X), or vector control were subjected to immunoblotting analysis directly (left) or after immunoprecipitation with anti-Flag antibody conjugated agarose beads (right). (I) Defective IκBα/NFκB signaling in patient-derived B cells can be reverted by full-length (long) but not mutant IKKβ(R286X). EBV-transformed B cells of a sibling and patients stably infected with retrovirus expressing WT IKKβ or with control vector were rested in phosphate-buffered saline at 37°C for 30 minutes, followed by PMA stimulation for 10 and 20 minutes. Immunoblotting analysis of cytosolic fractions (top) or nuclear extracts (bottom) with indicated antibodies. (J) Decreased expansion of patient-derived B cells can be corrected by full-length but not mutant IKKβ. *P < .05; **P < .01; ***P < .001 determined by Student t test.