Fig. 2

ATP promotes Paxillin phosphorylation and Paxillin-NLRP3 interaction. a–d THP-1 cells stably expressing control lentivirus or Paxillin lentivirus were generated and differentiated into macrophages, which were then treated with ATP (5 mM) or Nigericin (2 μM) for 2 h (a, b) and treated with DMSO, YVAD (50 μM), Glybenclamide (25 μg/ml), A438079 (10 μM), AZ10606120 (10 μM), and BAPTA-AM (30 μM) for 1 h before the treatment with ATP (5 mM) for 2 h (c, d). IL-1β levels in supernatants were determined by ELISA (a, c). Mature IL-1β (p17) and cleaved Casp-1 (p22/p20) in supernatants or Paxillin, pro-IL-1β, and pro-Casp-1 in lysates were determined by Western blot (b, d). e TPA-differentiated THP-1 macrophages were treated with DMSO or ATP (5 mM) for 2 h. Lysates were prepared and subjected to IP (top) or subjected to Western blot (as input) (bottom). f BMDM cells prepared from C57BL/6 mice bone marrow were treated with LPS (1 μg/ml) for 6 h, and the primed BMDMs were stimulated by DMSO or ATP (5 mM) for 30 min. Lysates were prepared and subjected to IP (top) or subjected to Western blot (as input) (bottom). g, h TPA-differentiated THP-1 macrophages were treated with ATP (5 mM) for 30, 60, and 120 min (g). LPS-primed BMDMs were stimulated by ATP (5 mM) for 5, 15, and 30 min (h). The protein level of p-Paxillin(Y118), p-Paxillin(Y31), Paxillin, and GAPDH was determined by Western blot. i HEK293T cells were co-transfected with pFlag-NLRP3 and pHA-Vector, pHA-Paxillin, pHA-Paxillin-(Y31A), and pHA-Paxillin-(Y118A). Lysates were prepared and subjected to IP (top) or subjected to Western blot (as input) (bottom). j TPA-differentiated THP-1 macrophages were treated with DMSO, A438079 (10 μM), and AZ10606120 (10 μM) for 1 h before the treatment with ATP (5 mM) for 2 h. The protein level of p-Paxillin(Y118), p-Paxillin (Y31), Paxillin, and GAPDH was determined by Western blot. Data shown are means ± SEMs; ***p < 0.0001. ns, no significance. Densitometry of the blots was measured by ImageJ