P38 directly phosphorylates human NLRP1 in the linker region, which is critical for activation by the ribotoxic stress response. (A) HEK^ASC-based cell lines constitutively expressing human NLRP1 or the indicated murine Nlrp1b alleles were treated with DMSO, 30 µM Tal, or 15 µM Aniso for the indicated times. Cells with ASC-EGFP specks were quantified by flow cytometry as described in Fig. 1. (B) 0.2 µM activated p38α was incubated with 7 µM MBP-NLRP1, MBP-NLRP1^NACHT-LRR (aa 230–990), or no substrate (⦰) at 30°C for 30 min in kinase buffer containing [³²P]-γ-ATP. Protein-associated ³²P was quantified by liquid scintillation counting. (C) Scheme of generated HEK^ASC-based cell lines constitutively expressing human NLRP1 mutants. The number of mutated amino acids in the PYD or linker is indicated to the left of the molecule. (D and E) HEK^ASC cell lines expressing the NLRP1 mutants described in C were stimulated and analyzed as in A (D), or transiently transfected with expression vectors for FLAG-tagged (VHL)-VHH for 20 h (E) and analyzed for FLAG expression and ASC-EGFP specks (in FLAG-positive cells, except for LF only controls). Data represents average values (with individual data points) from three independent experiments ± SEM (A, D, E) or SD (B).