Targeted RNAi knockdown of Btk protein results in decreased TNF, not IL-6, production in human macrophages after LPS stimulation. Peripheral blood monocytes were transfected with increasing doses of targeting and control siRNA oligonucleotides and differentiated in the presence of 100 ng/mL of M-CSF in culture for 4 days. (A) The expression of Btk was assessed by Western blotting. Densitometry units (mean ± SEM) for 4 separate donors are shown normalized to untreated controls. (B) For analysis of cytokine expression, siRNA-transfected M-CSF macrophages were treated with LPS (10 ng/mL) for 18 hours and supernatants assessed for TNF and IL-6 levels by ELISA. Values are shown as mean plus or minus SEM for 4 separate donors normalized to LPS only controls (**P< .01). (C) PBMCs were prepared from XLA and normal male donors (age range, 17–46 years) as described in “Isolation and culture of PBMCs from XLA patients and control donors.” Cells were left undifferentiated (XLA ■, normal ●) or cultured in the presence of M-CSF (100 ng/mL) for 4 days (XLA □, normal ○) and then stimulated with LPS (10 ng/mL) or Pam3C-SK4 (100 ng/mL). Cytokine production was assessed by ELISA 18 hours after stimulation. Each data point shown represents a single donor (n.s., not significant). (D) Bmx and Tec protein expression in matched undifferentiated and M-CSF-treated XLA PBMCs, normal PBMCs, and blood monocytes was assessed by Western blotting. Blots are representative of 4 separate donors for each matched cell population. Statistical significance was assessed using one-way ANOVA and Bonferroni multiple comparison test.