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. 2019 Nov 28;10:2791. doi: 10.3389/fimmu.2019.02791

Figure 6.

Figure 6

VA RNAI blocks ASC phosphorylation and oligomerization. HEK293 cells stably expressing YFP-ASC and co-transfected with plasmids expressing Flag-tagged PKR and a GFP-tagged NLRP3 fusion protein were infected with Ad WT or Ad ΔVAI and the cell lysates immunoprecipitated with anti-Flag antibodies. (A) Western blot analysis of coprecipitated NLRP3 and PKR using anti-GFP and anti-Flag-antibodies, respectively demonstrating a VA RNAI-independent interaction between NLRP3 and PKR. (B) Western blot analysis of coprecipitated ASC and PKR using anti-YFP and anti-Flag-antibodies, respectively, demonstrating a VA RNAI-dependent inhibition of the interaction between ASC and PKR. (C) Western blot analysis of phosphorylated ASC using a phospho-Tyr146-specific antibody demonstrating a VA RNAI-dependent inhibition of ASC phosphorylation. (D) Triton-X100 insoluble THP-1 cell pellets cross-linked with 4 mM disuccinimidyl suberate to preserve ASC oligomers. Samples dissolved in SDS sample buffer were subjected to Western blot analysis to detect ASC demonstrating a VA RNAI-dependent inhibition of ASC oliogmerization. The data ratio was calculated as the sum of oligomers and dimers vs. monomers.