Activation of NLRP1 by ubiquitination of NLRP1 ^PYD is independent of p38. (A) Alignment of NLRP1^PYD nanobodies (VHHs) with indicated complementarity determining regions (CDRs). (B) Binding of indicated concentration of HA-tagged VHHs to immobilized GST-NLRP1^PYD or control protein GST was quantified by ELISA. (C) Experimental setup and scheme of VHH-mediated ubiquitination of NLRP1. HEK^NLRP1+ASC and HEK^NLRP3+ASC cells were transiently transfected with expression vectors for VHL-VHH fusions; N/TERT-1 cells were transduced with lentiviral vectors encoding C1C-EGFP and (VHL-)VHH controlled by a bidirectional dox-inducible promoter. (D and E) HEK^NLRP1+ASC and HEK^NLRP3+ASC cells expressing the indicated FLAG-tagged VHL-VHH fusions or HA-tagged VHHs alone, where indicated in the presence of 10 µM Dora, 1 μM MLN4, 1 μM PF, 200 nM ISRIB, 3 µM Jnk, or DMSO, were analyzed for FLAG expression and ASC-EGFP specks 20 h after transfection as described in Fig. 1. Quantification of specks was limited to FLAG-positive cells in E. (F–I) N/TERT-1 cells inducibly expressing C1C-EGFP as well as VHL-VHH or VHH alone were treated with 1 µg/ml dox for the indicated time, where indicated in the presence of 10 µM Dora, 1 μM MLN7, 1 μM MLN4, 1 µM MG-132, 1 μM PF, 200 nM ISRIB, 3 µM Jnk, or DMSO. Expression of C1C-EGFP and (VHL-)VHH was induced in presence of 100 µM VX for analysis of speck assembly in C1C-EGFP–positive cells by flow cytometry (F and G), or in the absence or presence of VX as indicated for analysis of cell death by LDH release or IL-β secretion by HTRF (H and I). (G) The indicated N/TERT-1 keratinocytes cell lines described above were stimulated with 30 µM Tal in the absence of dox and analyzed for LDH release and IL-1β secretion as above. Data represents average values (with individual data points) from three independent experiments ± SEM.