Fig. 5
Blocking Tyr136 phosphorylation promotes aggregation and amyloid fibril formation of αSyn. a WT r-αSyn or Y136A r-αSyn after 0–40 h of incubation with CK2 and ATP was analyzed by SDS-PAGE followed by immunoblotting with anti-αSyn antibody D119. Molecular mass markers are indicated in kDa on the left side of each panel. Arrows indicate the top of the gel. b Intensity ratios (%) of immunoreactive > 250-kDa αSyn after 8 h of incubation were quantified in seven independent experiments. Data are presented as means ± standard deviation. Statistical significance was determined using the 2-tailed Student’s t test. ***P < 0.001 vs. WT r-αSyn. c ThT assays were performed in reactions with WT r-αSyn in the presence (WTCK2+ATP) or absence (WT) of CK2 and ATP or in reactions with Y136A r-αSyn in the presence (Y136ACK2+ATP) or absence (Y136A) of CK2 and ATP. The results show the kinetics of ThT fluorescence from one representative of six replicate wells for each condition. d The end products from reactions with WTCK2+ATP or Y136ACK2+ATP were examined by TEM. Bars, 100 nm. Values of e maximal fluorescence intensities and f lag phase obtained in six individual wells in ThT assay are plotted. Data are presented as means ± standard deviation. Statistical significance was determined using one-way ANOVA followed by Tukey–Kramer test. *P < 0.05, **P < 0.01