Figure 3.

The role of Eri in NLRP3 inflammasome assembly. (A) THP-1 cells were co-transfected with the indicated plasmid for 36 h, then treated LPS (1 µg/ml) for 6 h and ATP (2.5 mM for 30 min). IL-1β and IL-18 protein levels were determined by ELISA. (B) HEK293T cells were transfected with Flag-tagged NLRP3 (Flag-NLRP3) or Myc-tagged MEK7 (Myc-MEK7) for 36 h. Then, cells were treated with Eri (5 nM) for 6 h. Co-IP and immunoblot analysis were performed with the indicated antibodies. (C, D) Experiments were performed as described in (B), except Myc-ASC (C) or Myc-ASC and Flag-Casp-1 (D) were used. (E, F) Purified GST-NLRP3, His-MEK7, and His-ASC were mixed (3 nM) in various combinations as indicated, and incubated with Eri (3 nM). The solution was immunoprecipitated with indicated antibody and analyzed using western blotting. (G) THP-1 cells were treated with Nigericin (2 μM for 2 h) and Eri (5 nM for 6 h). Co-IP and immunoblot analysis were performed with the indicated antibodies. All experiments were repeated at least three times. Bar graphs present means ± SD (**P < 0.01; n.s., not significant).