Fig. 6

BMAA activates neuronal innate immunity. In primary mouse cortical neurons pre-treated with 1 μM CCCP, 3 mM BMAA, and 1 μg/μl LPS for 48 h or with 3 mM BMAA for6, 24 and 48 h, we evaluated a changes in protein levels of TLR4 and b TLR 3 by western blotting. Representative immunoblot and densitometric analysis was performed by the loading of equal amounts of protein corrected with β-actin. Inflammasome activation was determined by the quantification of c cytosolic IkBα protein levels which were determined by western blotting. Representative immunoblot and densitometric analysis was performed by loading equal amounts of protein corrected with β-actin. d p65 NFκB was detected in enriched nuclear fractions by western blotting. Representative immunoblot and densitometric analysis was performed by the loading of equal amounts of protein corrected with TATA-binding protein. e Cytosolic NLRP3 protein levels were evaluated by western blotting. Representative immunoblot and densitometric analysis was performed by loading equal amounts of protein corrected against β-actin. f Cytosolic pro-IL-1β protein levels analyzed by western blotting. Representative immunoblot and densitometric analysis was performed by the loading of equal amounts of protein corrected with β-actin. g p62 flux was evaluated by western blotting after 4-h incubation with or without NH₄Cl plus leupeptin. Representative immunoblot and densitometric analysis was performed by the loading of equal amounts of protein corrected with β-actin. h Caspase 1-like activity was determined by spectrophotometry at 450 nm, and i quantification of IL-1β levels in cytosolic fractions and j in supernatants was determined using ELISA. Data represent mean ± SEM values derived from four to six independent determinations and are expressed relative to untreated neurons. The statistical significance was determined using one-way ANOVA followed by Dunnett’s test *p < 0.05 and **p < 0.01 when compared to untreated neurons