P38 activation is sufficient for NLRP1 activation. (A–F) HEK^NLRP1+ASC or HEK^NLRP3+ASC cells were transiently transfected with empty vector (A), expression vectors for p38α (B), p38α T180A Y182A (p38α AA; C), p38β (D), p38γ (E), or p38δ (F) in combination with empty vector (⦰) or the indicated expression vectors for MKK1, MKK3, MKK3 S218E T222E (MKK3 EE), MKK3 S218A T222A (MKK3 AA), MKK4, MKK5, or MKK6 for 20 h. Where indicated, cells were treated with 10 µM Dora. Cells with ASC-EGFP specks were quantified by flow cytometry as described in Fig. 1. (G and H) HEK^NLRP1+ASC or HEK^NLRP3+ASC cells were transiently transfected with expression vectors for FLAG-tagged catalytic domain of TAOK2 (TAOK2 CD), where indicated in the presence of 10 µM Dora, 1 μM MLN4, 1 μM PF, 200 nM ISRIB, 3 µM Jnk, 100 nM ZAKi, or DMSO, and analyzed for FLAG expression and ASC-EGFP specks in FLAG-positive cells 20 h after transfection. Specks of total ASC-EGFP–positive cells were quantified in case of LF only controls. Data represents average values (with individual data points) from three independent experiments ± SEM.