Fig. 6.

Effect of paraquat on calpain activation and accumulation of CT α-syn. a, b OB protein extracts from non-transgenic (WT), A30P and paraquat-treated A30P+P and dox-treated control (A30P+D+P, A30P+D) mice were loaded on a SDS-PAGE or c native PAGE. a, c Antibodies specific for calpain 1 and calpastatin (CAST), its specific inhibitor, were used. b Quantification of immunoblots shows that A30P+P mice present increased level of calpain-1 and d complex formation of CAST. The data are expressed as mean ± SEM. **p < 0.01 vs. Ntg, ^§p < 0.05 vs. A30P, ⁺p < 0.05 vs. A30P+D. e Cell biological analysis of SH-SY5Y human neuroblastoma cells treated with increasing concentration of paraquat. Cell viability was assessed at 48 and 72 h after treatment with paraquat, with and without 10 μM of the calpain inhibitor calpeptin, using LDH assay. f Non-cytotoxic concentration (150 μM) was used to analyze paraquat-induced activation of calpain and g, h a correlating formation of CT α-syn. f Increase in calpain activity in SH-SY5Y cells exposed to 50, 150 μM of paraquat was reduced by parallel treatment with calpeptin (10 μM) for 72 h. g Representative immunoblot of SH-SY5Y with 105 antibody against human full-length and CT α-syn shows increase of endogenous full-length and CT α-syn. Infecting cells with lentiviral human α-syn (+LV Syn) was used as a positive control and showed expected relative strong reactivity of 105 against full-length and CT α-syn. Calpeptin, a specific inhibitor (Inh) of calpain treatment reduced level of FL α-syn and CT α-syn. h Correlation between (fold) increase of CT α-syn and (fold) increase of calpain activity dependent on concentration of paraquat. Immunoblots are highlighted by separating boxes for clarity of display. The data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT; ^§p < 0.05, ^§§p < 0.01 vs. A30P; ⁺p < 0.05, A30P+calp vs. A30P+P+calp