XIAP deficiency impairs TCR-, LPA-, and EGF-stimulated cytokine production and NF-κB activation. (A) Diminished IFN-γ production in Xiap^−/− T cells. Purified T cells from normal littermate control and Xiap^−/− mice were activated with plate-bound anti-CD3 (5 μg/mL), and the IFN-γ secretion was determined 48 hours later. (B-C) Reduced CD3-induced NF-κB activation in Xiap^−/− T cells. Control and Xiap^−/− T cells were activated with anti-CD3, and (B) total cell lysates and (C) nuclear extracts were prepared at the indicated time points. (B) IKK phosphorylation in total cell lysates and (C) the presence of p65 in nuclear extracts were determined by immunoblotting. HDAC2 was the internal control of the nuclear extract, whereas glyceraldehyde-3-phosphate dehydrogenase was the marker of the cytosolic extract. (D-E) Diminished LPA-stimulated IL-6 production and NF-κB activation in Xiap-deficient macrophages. Peritoneal macrophages were isolated from WT and Xiap^−/− mice primed with thioglycolate and were stimulated with LPA (10 μM). (E) Nuclear extracts were prepared at 30 and 60 minutes, and the presence of p65 as determined. (D) IL-6 was quantitated by ELISA 16 hours later. (F-G) XIAP knockdown decreased EGF-induced NF-κB activation. Control siRNA or XIAP-specific siRNA was transfected into HepG2 cells by electroporation. HepG2 cells were plated 16 hours after transfection and treated with EGF (20 ng/mL) after another 24 hours. Total cell lysates and nuclear extracts were prepared at the indicated time points. (F) EGF-induced IκBα phosphorylation and (G) p65 nuclear translocation were measured in total cell lysates and nuclear extracts, respectively. *P < .05, **P < .01, and ***P < .001 for paired Student t test. (A,D) Each data point represents mean of triplicate determinations in a single experiment, and (A-G) each experiment was repeated 3 times with similar results.