Fig. 6

USP13 is essential for Paxillin-mediated NLRP3 deubiquitination upon ATP treatment. a HEK293T cells were co-transfected with pHA-Paxillin and pFlag-BRCC3, pFlag-EIF3S5, pFlag-USP13, or pFlag-OTUB1. Lysates were prepared and subjected to IP (top) or subjected to Western blot (as input) (bottom). b Hela cells stably expressing sh-USP13 or sh-EIF3S5 were generated and analyzed. c The stable Hela cells were co-transfected with pFlag-NLRP3, pHA-UB, or pMyc-Paxillin. Lysates were prepared and subjected to denature-IP (top) or subjected to Western blot (bottom). d, e HEK293T cells were transfected with pFlag-USP13 and pHA-Paxillin. f, g HEK293T cells were transfected with pFlag-USP13 and pHA-NLRP3. Lysates were prepared and subjected to IP (top) or subjected to Western blot (bottom) (d–g). h HEK293T cells were co-transfected with pFlag-USP13 and pHA-NLRP3 or pHA-NLRP3 mutants. i The diagrams of USP13 and its mutants, as indicated. j HEK293T cells were co-transfected with pHA-NLRP3 and pFlag-USP13 or pFlag-USP13 mutants. k HEK293T cells were co-transfected with pHA-Paxillin and pFlag-USP13 or pFlag-USP13 mutants. l HEK293T cells were co-transfected with pFlag-USP13 and pHA-Paxillin or pHA-Paxillin mutants. Lysates were prepared and subjected to IP (top) or subjected to Western blot (bottom) (h, j–l). m HEK293T cells stably expressing sh-USP13 were generated. The stable cells were co-transfected with pFlag-NLRP3 and pHA-UB. n The stable HEK293T cells were co-transfected with pFlag-NLRP3 or pHA-UB with or without Spautin-1 (20 μM) for 6 h. o The stable HEK293T cells were co-transfected with pFlag-NLRP3, pHA-UB, or pMyc-Paxillin with or without Spautin-1 (20 μM) for 6 h. Lysates were prepared and subjected to denature-IP (top) or subjected to Western blot (bottom) (m–o). p, q TPA-differentiated THP-1 macrophages were treated with ATP (5 mM) for 2 h with/without Spautin-1 (5, 10, and 20 μM). IL-1β in supernatants was determined by ELISA (p). Mature IL-1β(p17) and cleaved Caspase-1(p22/p20) in supernatants and Paxillin, pro-IL-1β, and pro-Casp-1 in lysates were determined by Western blot (q). Data shown are means ± SEMs; ***p < 0.0001