A, Western blot for TRAF3 in PBMCs from the patient (P) and healthy volunteers (V1 and V2). PBMCs were isolated from the patient and healthy volunteers and were unstimulated (RPMI) or stimulated with LPS or Poly I:C and showed diminished TRAF3 expression in patient cells. Data representative of 2 independent experiments are shown. B, Empty vector, wild-type (WT TRAF3), and mutant (R338W TRAF3) plasmids were transfected in HEK 293FT cells. Twenty-four hours after transfection, cells were stimulated with LPS or Poly I:C for 24 hours. Western blot analysis demonstrated diminished TRAF3 expression in transfected R338W cells compared with WT. Data are representative of 2 independent experiments. C, Empty vector, wild-type (WT TRAF3), mutant (R338W TRAF3) plasmids were transfected in RAW264.7 cells. The cells were also transfected with equal amounts of both plasmids (WT + R338W TRAF3). Western blot was performed on the cells 48 hours after transfection. Diminished TRAF3 expression was observed in R338W and WT + R338W TRAF3–transfected cells compared with WT. Data representative of 2 independent experiments are shown. D, Empty vector, wild-type (WT TRAF3), and mutant (R338W TRAF3) plasmids were transfected in RAW264.7 cells. The transfected cells were stimulated after 24 hours with LPS or NTM. ELISA for mouse IL-6 and TNF-α was performed on the cell supernatants 24 hours after stimulation. Data were pooled from 2 experiments. *P < .05 based on Mann-Whitney U test. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IL-6, interleukin 6; LPS, lipopolysaccharide; NTM, nontuberculous mycobacteria; PBMC, peripheral blood mononuclear cell; TNF-α, tumor necrosis factor alpha; WT, wild-type.