Fig. 5.

ROS activates MINK1 kinase to prime the activation of NLRP3. A LPS-primed BMDMs from Mink1^−/− and WT mice were stimulated with ATP, and total ROS and mitochondrial ROS levels were measured by ROS and MitoSOX incorporation using flow cytometry. B, C LPS-primed BMDMs from Mink1^−/− and WT mice were treated with NAC (3 mM) 30 min before imiquimod (a small-molecule ligand of Toll-like receptor-7 (TLR7)) stimulation. Supernatants (SN) and cell extracts (lysate) were analyzed using immunoblotting (C), and supernatants were analyzed using ELISA for IL-1β (B). D–F LPS-primed BMDMs were treated with NAC 30 min before imiquimod stimulation, MINK1 was immunoprecipitated, and in vitro kinase assays were performed with myelin basic protein as the substrate in the presence of ATP-γ-S. The reaction products were immunoblotted with an antithiophosphate ester antibody (D). Supernatants were analyzed using ELISA for IL-1β (E), and the fold change of kinase activity was quantified and is presented in F. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t test was used for F, and ANOVA was used for B and E. The flow cytometry, ELISA, and western blot results are representative of three independent experiments