NLRP1 activation by ribotoxic stress response relies on the ubiquitination machinery and proteasomes. (A–E) HEK^NLRP1+ASC (A and E), N/TERT-1^C1C-EGFP (B), or HEK^NLRP3+ASC (C and D) were stimulated with DMSO, 1 mM CHX (A and B), 20 µg/ml Blasti (A and B), 200 ng/ml LPS followed by 10 µM Nig (C and D), 10 mM azide and 50 mM 2-DG (E), or 1.5 mM H₂O₂ (E) for the indicated times. Cells were stimulated in the presence of 10 µM Dora, 1 μM MLN4, 1 μM PF, 200 nM ISRIB, 3 µM Jnk, 1 μM MLN7, 20 μM SB, 20 µM BeMeEs, 1 µM MG-132, 1 µM Borte, or DMSO as indicated. Note that LPS + Nig stimulation was performed towards the end of inhibitor treatment to evaluate NLRP3 responses after 6 h (C) or 20 h (D) in the presence of the indicated inhibitors. Experiments were done in parallel to data shown in Fig. 3, A and C. Specking cells were quantified by flow cytometry as in Fig. 1, and N/TERT-1 cells were always stimulated in the presence of 100 µM VX. Data represents average values (with individual data points) from three or four independent experiments ± SEM.