Fig. 4

Role of Ca²⁺ and CaM in mitochondria complex I inhibitor-induced Ser129-phosphorylation of α-syn. As vehicle control, cells were treated with DMSO at the same final concentration as reagents used. Cell lysates (10 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a, b Effect of intracellular Ca²⁺ chelator BAPTA-AM (a) or extracelluar Ca²⁺ chelator EGTA (b) on MPP⁺-induced Ser129-phosphorylation. Wt-aS/SH #4 cells were incubated in media containing 1 mM MPP⁺ and the indicated concentrations of BAPTA-AM or EGTA for 16 h. c, d Effect of BAPTA-AM (c) and EGTA (d) on rotenone-induced Ser129-phosphorylation. Wt-aS/SH #4 cells were incubated in media containing 5 μM rotenone and the indicated concentrations of BAPTA-AM or EGTA for 16 h. e, f Effect of CaM inhibitor W-7 on MPP⁺ (e)- or rotenone (f)-induced Ser129-phosphorylation. Cells were incubated in media containing 1 mM MPP⁺ or 5 μM rotenone with W-7 at the indicated concentrations for 16 h. Representative blots are shown. In the graphs of a to f, relative band intensities of Ser129-phosphorylated α-syn and total α-syn were normalized to those of β-actin. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction (*, P < 0.05; **, P < 0.01)