Figure 5. FBXO11^−/− macrophages exhibit higher IL-1β production.

(A, B) WT, FBXO11^−/−, and (B) C5aR1^−/− macrophages were primed with LPS (100 ng/ml) for 3 h before PVL (62.5 ng/ml), LukAB (15.6 ng/ml), or nigericin (10 μM) treatment for 2 h. Il-1β in culture supernatants were determined by ELISA. Mean ± SEM of three independent biological replicates shown. ns = not significant,; * = P < 0.05, *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA followed by Sidak’s multiple comparisons test. Abbreviation: Nig, nigericin. (C) Western blot analysis of pro-IL-1β (34 kD) and cleaved IL-1β (17 kD) in cell lysates and supernatants of WT and FBXO11^−/− macrophages treated with LPS and/or toxins. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Abbreviation: S, short exposure; L, long exposure. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Western blot analysis of pro-IL-1β in whole cell lysates of unprimed THP1 cells. Monocyte (Mono) was differentiated with PMA, and macrophages were cultured in PMA-free media for the indicated amount of time. Protein abundance was normalised to β-actin and represented as fold change compared with monocytes. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01, *** = P < 0.001; by two-way ANOVA with Sidak’s multiple comparisons test. (E) Western blot analysis of NLRP3 in whole-cell lysates of WT and FBXO11^−/− macrophage. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by one-way ANOVA followed by Dunnett’s multiple comparison test. (F) Western blot analysis of pro-IL-1β and MCL-1 in WT and FBXO11^−/− macrophages. Cells were primed with LPS (100 ng/ml) for 2 h, with MG132 (20 μM) and Q-VD-OPh (20 μM) added in the last 30 min alongside. Cells were then treated with CHX (20 μg/ml) with or without MG132 for the indicated amount of time before cell lysate collection. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (G) Western blot analysis of pro-IL-1β in WT and FBXO11^−/− macrophage cell lysates and TUBE-isolated ubiquitinated proteins. WT* indicates agarose beads only as control. (H) qRT-PCR analysis of relative IL-1β mRNA level. The mRNA levels were normalised to the control values of GAPDH. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test.

Source data are available for this figure.