Serine 107 in the linker region is critical for p38-dependent NLRP1 activation. (A) Overview of mutants of NLRP1 phosphorylation sites. Phosphosite T41 was described previously, while all other phosphosites were detected in immunoprecipitated NLRP1-HA after stimulation with 15 μM Aniso for 1 h. (B and C) HEK^ASC-based cell lines constitutively expressing human NLRP1 or the indicated mutants were treated with DMSO, 30 µM Tal, or 15 µM Aniso for the indicated times and analyzed for specks as described in Fig. 1 (B), or transiently transfected with expression vectors for FLAG-tagged VHL-VHH for 20 h (C) and analyzed for FLAG expression and ASC-EGFP specks (in FLAG-positive cells, except for LF only controls). (D) Monoclonal NLRP1 knockout N/TERT-1^C1C-EGFP cells reconstituted with dox-inducible NLRP1 or the indicated NLRP1 mutants were treated with dox for 6h. Cells were subsequently stimulated with DMSO, 30 µM Tal or 15 µM Aniso, or infected with SFV at an MOI of 5 for 20 h in the presence of 100 μM VX. Infected cells were stained with antibodies for dsRNA and infection and speck assembly (in infected cells in case of SFV) were quantified by flow cytometry. Data represents average values (with individual data points) from three or four independent experiments ± SEM.