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. 2020 Mar 18;16(3):e1008335. doi: 10.1371/journal.ppat.1008335

Fig 6. STING deubiquitinates NLRP3 to activate the NLRP3 inflammasome.

Fig 6

(A–E) Hela cells were co-transfected with pFlag-NLRP3, pHA-Ubiquitin, and pMyc-STING (A), with pFlag-NLRP3, pHA-Ubiquitin, pHA-Ubiquitin mutations (K48R), pHA-Ubiquitin mutations (K63R) and pMyc-STING (B), with pFlag-NLRP3, pHA-Ubiquitin, pHA-Ubiquitin mutations(K48O), pHA-Ubiquitin mutations(K63O), and pMyc-STING (C), with pFlag-NLRP3 and pHA-Ubiquitin and then infected with HSV-1(MOI = 1) for 2 and 4 h (D), and transfected with HSV120 (3 μg/ml) for 2 and 4 h (E). Cell lysates were prepared and subjected to denature-IP using anti-Flag antibody and then analyzed by immunoblotting using an anti-HA or anti-Flag antibody (top) or subjected directly to Western blot using an anti-Flag, anti-HA or anti-Myc antibody (as input) (bottom). (F–I) TPA-differentiated THP-1 macrophages were infected with HSV-1 (MOI = 1) for 2 and 4 h (F), and transfected with HSV120 (3 μg/ml) for 2, 4 and 6 h (G). Mice primary MEFs were infected with HSV-1 (MOI = 1) for 2 h (H). THP-1 cells stably expressing sh-NC or sh-STING were generated and differentiated to macrophages and then infected with HSV-1 (MOI = 1) for 4 h (I). Cell lysates were prepared and subjected to denature-IP using anti-NLRP3 antibody and then analyzed by immunoblotting using an anti-Ubiquitin, anti-K48-Ubiquitin, anti-K63-Ubiquitin or anti-NLRP3 antibody (top) (F) or using anti-NLRP3 antibody (G, H and I), or subjected directly to Western blot using an anti- Ubiquitin or anti-NLRP3 antibody (as input) (bottom).