Fig 1: UAF1/USP12 and UAF1/USP46 complexes promote NLRP3 transcription.a RT-PCR analysis of Il1b, Il6 and Tnf mRNA expression in Ctrl siRNA−, Usp1 siRNA−, Usp12 siRNA−, or Usp46 siRNA-transfected mouse peritoneal macrophages, followed by stimulation with LPS for 2 h (mean ± SEM, two-tailed t-test Usp1 siRNA, Usp12 siRNA and Usp46 siRNA vs. Ctrl siRNA, left panel: ns = 0.8310, *P = 0.0135, **P < 0.0001 in sequence, middle panel: ns = 0.4945, **P = 0.0006, 0.0003 in sequence, right panel: ns = 0.8037, **P = 0.0005, 0.0007 in sequence; n = 3 independent experiments). b Analysis of NF-κB reporter activation using a Luciferase assay in HEK293T cells transiently transfected with the indicated plasmids (mean ± SEM, two-tailed t-test USP1, USP12 and USP46 plasmids vs. empty control plasmid, ns = 0.1774, *P = 0.0106, 0.0298 in sequence; n = 6 independent experiments). c, d Western blot analysis of p65 or p-p65 in Ctrl siRNA-, Usp12 siRNA-, or Usp46 siRNA-transfected mouse peritoneal macrophages followed by stimulation with LPS for the indicated time periods. e Western blot analysis of p65 in WT or Uaf1 deficient mouse peritoneal macrophages after stimulation with LPS for the indicated time periods. f Cell lysates of mouse peritoneal macrophages stimulated with LPS for the indicated time periods were subjected to immunoprecipitation (IP) with anti-p65, followed by western blot analysis with antibodies to UAF1, USP1, USP12, USP46, or p65. Proteins from a whole-cell lysate were used as a positive control (Input). g Cell lysates of HEK293T cells transiently transfected with Myc-p65, Flag-USP1, Flag-USP12, Flag-USP46, and Flag-UAF1 were subjected to IP using Myc antibody, followed by western blot analysis with anti-Myc or anti-Flag. h, j Cell lysates of HEK293T cells transiently transfected with Myc-p65, HA-Ub, or HA-tagged K48-linked ubiquitin (HA-K48-Ub), Flag-USP12, USP46, and Flag-UAF1 were subjected to IP with Myc antibody, followed by western blot analysis with anti-HA. i Western blot analysis of extracts from LPS-stimulated WT or Uaf1CKO macrophages, followed by IP with anti-p65, and subsequent probing with anti-Ub. US, unstimulated. Similar results were obtained from three independent experiments.