Fig. 3

CSE decreases the half-life of the NLRP3 protein. a THP1 cells were incubated with or without CSE at 80 μg/mL for 18 h prior to CHX exposure at 40 μg/mL for the indicated time periods. Cells were collected and assayed for NLRP3 and β-actin (loading control) by immunoblotting for a half-life study. Representative images are shown. b Densitometric plots of NLRP3 protein decay versus time of CHX exposure with best fit lines, depicting the pooled data mean ± S.D. of three independent experiments. c Primary human macrophages from peripheral blood were transfected with 4 μg of either WT NLRP3-V5 or the point mutant K689R NLRP3-V5 plasmid. Following transfection, the cells were incubated with or without CSE at 120 μg/mL for 21 h, prior to CHX exposure at 40 μg/mL at different time points for a half-life study. Cells were collected and assayed for NLRP3-V5 and β-actin by immunoblotting. d Densitometric plots of adjusted NLRP3 protein decay over time under different conditions were best fitted. The half-life of WT NLRP3 protein was reduced with CSE exposure, while a K689R NLRP3 mutant was not. Data are mean ± S.D. of two independent experiments