Figure 6

The effect of SB exposure on α-Syn levels in TetOff-induced cultures is minimal. One day after the seeding of 3D5 cells RA was added to the cultures to elicit differentiation for 10 ds. Two subsets of these cultures were induced after 5 ds of differentiation to express α-Syn for 5 ds. Another set was induced after 1 d of differentiation to express α-Syn for 9 days. One set of the 5 d-induced cultures was exposed to SB for 24 hs before cell harvesting (labeled as SB-5D). Cultures with 5 and 9 ds of induced α-Syn expression and without SB exposure are marked as Con-5D and Con-9D, respectively. Cell lysates containing the same amount of proteins from different samples derived from 3 independent experiments were used for dot blotting with antibodies to α-Syn and GAPDH, or Western blotting. Quantitative analyses were carried out on dot blots to determine the ratio of α-Syn to GAPDH. When the ratio derived from Con-5D dot blots was set as 1, the relative amount of α-Syn to GAPDH is 0.97 ± 0.09 (average ± standard error of mean) and 1.58 ± 0.06 for SB-5D and Con-9D preparations, respectively. Based on Student's t-test, there are no significant differences (P > 0.8) between Con-5D and SB-5D. In comparison, there are significant differences (P < 0.001) between Con-9D and SB-5D or Con-5D. Since α-Syn oligomers were readily detected only in SB-5D cultures, their production/accumulation is not caused by an increase in the expression of α-Syn alone.