Fig. 3 |. Gsdmd cleavage requires the protease activity of papain.

a, Immunoblot analysis of WCL and culture supernatants from MLE-12 cells treated with papain (5 μg) for 30 min, an E-64 pretreatment for 30 min before papain stimulation, and heat-inactivated papain (Ina-pap, 10 min for 100 °C) for 30 min. b, Immunoblot analysis of WCL and culture supernatants from MLE-12 cells treated as follows. Aspergillus oryzae: 0, 0.5, 5, 10, 50 (μl well⁻¹); HDM: 1, 10, 20, 50, 100 (μg well⁻¹); Bacillus licheniformis: 0.05, 0.1, 1, 5 (μg well⁻¹); Der p: 0.01, 0.025, 0.25, 1, 2.5 (μg well⁻¹); Der f: 0.01, 0.025, 0.25, 2.5 (μg well⁻¹). Aspergillus oryzae and HDM were stimulated for 30 min; Bacillus licheniformis, Der p and Der f were stimulated for 60 min. c, Immunoblot analysis of MLE-12 cells treated with ovalbumin (OVA, 0.1–100 μg well⁻¹) for 30 min, aluminum (Alum, 0.1–100 μg well⁻¹) for 30 min, FSL-1 (1–500 ng ml⁻¹ per well) for 6 h, palmitic acid (0.1–50 μM well⁻¹) for 6 h, and poly-L-arginine (cationic polypeptides, 2.5–125 μg well⁻¹) for 30 min. Red arrows indicate the neo-form of p40 NT-Gsdmd. Nonspecific fragments marked with an asterisk were not discussed in our work. a–c, Data are representative of three independent experiments.