EV71 3D Protein Binds with NLRP3 and Enhances the Assembly of Inflammasome Complex
Fig 2
EV71 activates IL-1β through regulating the components of NLRP3 inflammasome complex.
(A and B) HEK293T cells were transfected with plasmids encoding pro- IL-1β, Flag-pro-caspsase-1, Flag-NLRP3, and Flag-ASC. EV71 (MOI = 1 or 2) infected the transfected cells for 24 h. 5 mM ATP for 30 min as a positive control. Supernatants were analyzed by ELISA for IL-1β secretion (A). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific to pro-IL-1β, pro-caspase-1, EV71 3D protein and GAPDH (Lys) (B). (C and D) TPA-differentiated THP-1 cells with shRNA-mediated specific gene silencing were stimulated by 2 μM Nigericin for 2 h. Supernatants were analyzed by ELISA for IL-1β secretion (C). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific to IL-1β, Caspase-1, NLRP3, ASC, and GAPDH (Lys) (D). (E and F) TPA-differentiated THP-1 cells with shRNA-mediated knockdown of gene expression were infected by EV71 (MOI = 20) for 24 h. Supernatants were analyzed by ELISA for IL-1β secretion (E). Immunoblot analysis of the mature (p17) form of IL-1β in the supernatants (Sup). Cell lysates were normalized for protein content and analyzed by immunoblotting using antibodies specific to IL-1β, Caspase-1, NLRP3, ASC, EV71 3D protein, and GAPDH (Lys) (F). Data shown are means±SEM, **p<0.01,***p<0.0001.