Figure 4.

Eri associates with NLRP3. (A) Immunoblot analysis of binding complexes isolated from BMDMs extracts incubated with biotin-labeled Eri (Bio-Eri) or free Eri. (B) BMDMs extracts were incubated with Bio-Eri and different concentrations of free Ori. The total proteins (input) and pulled down proteins (PD) were immunoblotted as indicated. (C) Purified human GST-NLRP3 protein was incubated with indicated doses of Bio-Eri. Total proteins (input) and pulled down proteins (PD) were immunoblotted as indicated. (D) C57BL/6 mice were intraperitoneally injected with the indicated concentrations of Eri or Bio-Eri for 12 h. Then, mice were intraperitoneally injected with MSU (1 mg/mouse) for 6 h The total proteins (input) and pulled down proteins (PD) in peritoneal fluids were immunoblotted as indicated. (E) MST assay for the affinity between Ori and purified GST-NLRP3 protein. (F) HEK293T cells were transfected with indicated plasmids for 36 h. Immunoblot analysis of binding complexes isolated from cells extracts incubated with Bio-Eri. (G) Schematic diagram of the full-length and truncated constructs of NLRP3 (upper panel). HEK293T cells were transfected with indicated plasmids for 36 h. Immunoblot analysis of binding complexes isolated from BMDMs hextracts incubated with Bio-Eri (lower panel). (H) BMDMs were treated with LPS (1 µg/ml for 6 h) or Eri (5 nM for 15 min), and washed or no washed for three times. Then, cells treated with Nigericin (2 μM) for 2 h. IL-1β protein levels in supernatant were determined by ELISA. (I) BMDMs extracts were incubated with free Eri for 4 h before or after incubated with bio-Ori (1 μM) for 4 h. Total proteins (input) and pulled down proteins (PD) were immunoblotted as indicated. All experiments were repeated at least three times. Bar graphs present means ± SD (**P < 0.01).