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Fig. 2 | Acta Neuropathologica Communications

Fig. 2

From: Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates

Fig. 2

Effects of Ca2+ on Ser129-phosphorylation of α-syn. SH-SY5Y cell lines stably expressing wild-type α-syn (wt-aS/SH #4) were incubated in media containing 5 μM calcium ionophore A23187 except b. As vehicle control, cells were treated with DMSO at the same final concentration as reagents used. Cell lysates (15 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or anti-β-actin (AC-15) antibody. a Effect of A23187 incubation time on Ser129-phosphorylation. Cells were treated with A23187 for the indicated time points until 8 h. b Effect of A23187 concentrations on Ser129-phosphorylation. Cells were treated with A23187 at the indicated concentrations for 8 h. c, d Effect of extracellular Ca2+ chelator EGTA (c) or intracellular Ca2+ chelator BAPTA-AM (B-AM) (d) on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing 5 μM A23187 with the indicated concentrations of EGTA or BAPTA-AM for 4 h. e, f Effect of CaM inhibitor W-7 (e) or calmidazolium (Calm) (f) on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing A23187 with the indicated concentrations of W-7 or calmidazolium for 4 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 or Welch-ANOVA with Games-Howell post hoc test for unequal-variances (*, P < 0.05; **, P < 0.01)

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