Fig. 8

ACT001 suppressed NFκB/NLRP3 neuroinflammatory pathway by inhibiting the phosphorylation of AKT. (a-b) After co-treatment with indicated doses of ACT001 and 100 ng/ml LPS for 24 h, rat primary microglia cells (a) and BV2 cells (b) were subjected to immunoblot analysis for p-AKT/Total-AKT, p-IKKα/β/Total-IKKβ, p-NFκB/Total-NFκB, NLRP3, ASC, cleaved-Caspase-1/pro-Caspase-1, IL-1β/pro-IL-1β expression, and GAPDH was used as c ontrol for protein loading. c Schematic diagram of ACT001 probe pull-down experiment. d Chemical structure of ACT001-S-biotin probe (inactive) and ACT001-biotin probe (active) were shown as indicated. e Proteins pulled down by the probe in rat primary microglia cells and BV2 cells were detected through silver staining. Input referred to the whole protein lysates from these two cells; negative referred to ACT001-S-biotin probe solution; and positive referred to proteins pulled down by ACT001-biotin probe. f Proteins precipitated by ACT001-biotin probe or ACT001-S-biotin probe in rat primary microglia cells and BV2 cells were detected by western blotting using an anti-Total-AKT primary antibody