Figure 1. XIAP is required to repress LPS- and TNF-induced IL-1β secretion.

(a–f) WT and Xiap-deficient (x^−/−) macrophages were pre-incubated with or without (a–c) LPS (20 ng ml⁻¹) or (d–f) human Fc-TNF (100 ng ml⁻¹) for 2–3 h and cultured with or without IAP antagonists of differing IAP specificities (see g). After 24 h, cell supernatants were assayed for (a,d) IL-1β, (b,e) TNF and (c,f) IL-6 levels by ELISA. n=3 mice; Data are represented as mean+s.e.m., from one of three experiments. (g) Efficiency of functional XIAP antagonism by IAP antagonist compounds (+, high; −,low). (h) WT, cIAP1^−/− (c1^−/−), cIAP2^−/− (c2^−/−) and Xiap^−/− (x^−/−) BMDM were primed with LPS (20 ng ml⁻¹) for 3 h and cultured with the IAP antagonist LBW242 (20 μM) or alum (320 μg ml⁻¹) for a further 6 h. Secreted IL-1β was measured in supernatants by ELISA. n=3 mice; mean+s.e.m. (i) Yield of macrophages from WT and IAP mutant bone marrow after 6 days of culture with L929 cell conditioned media. n=3–6 mice per genotype, mean+s.e.m. (j–l) WT and IAP mutant macrophages were stimulated with LPS (20 ng ml⁻¹) for up to 24 h, and (j) IL-1β, (k) TNF and (l) IL-6 levels were assayed in supernatants by ELISA. n=3–4 mice, data are represented as mean+s.e.m., one of three experiments. (m) Yield of WT, c1^lox/loxx^−/−c2^−/−, c1^LysMcrex^−/−c2^−/− and c1^ERcrex^−/−c2^−/− bone marrow macrophages after 6 days of culture with L929 cell conditioned media. n=3–6 mice per genotype, mean+s.e.m. (n,o) WT, c1^lox/loxx^−/−c2^−/−, c1^LysMcrex^−/−c2^−/− and c1^ERcrex^−/−c2^−/− macrophages were pulsed for 16 h with 4′-hydroxy-tamoxifen (4HT 1000, nM) and then rested for 10 h prior to stimulation with or without LPS (50 ng ml⁻¹) for a further 8 h. (n) Secreted IL-1β was measured in supernatants by ELISA, n=3 mice per group; c1^LysMcrex^−/−c2^−/− (n=2), mean+s.d., one of three experiments, and (o) IL-1β and caspase-1 activation assayed by immunoblot of supernatants and lysates. Representative blot from the analysis of 4 c1^ERcrex^−/−c2^−/− mice. Full-size immunoblots are presented in Supplementary Fig. 9.