Figure 3. PARK9 expression affects intracellular and extracellular levels of α-syn. A, The α-syn ELISA standard curve plotted OD450 (on the y-axis) and α-syn protein concentration (on the x-axis). B, The human PARK9-expression levels in PARK9-silenced and PARK9-overexpressed H4 cells (n = 3, *p < 0.01). C, Top, Immunoblot analysis of α-syn expression in Scrb and PARK9 shRNA-treated H4 cells. Bottom, Quantification of α-syn expression level. After normalization to NSE, the expression of α-syn in PARK9-silenced H4 cells was divided by expression in Scrb shRNA-transfected cells (n = 4, *p < 0.03). D, Top, Immunoblot analysis of α-syn expression level in mock and PARK9 overexpressed H4 cells (n = 4, *p < 0.03). Bottom, Quantification analysis of α-syn expression level; (n = 3, *p < 0.03). Data were analyzed by Student t test (C, D). E, Representative images of mock transfected (right) and PARK9 overexpressed H4 cells. PARK9 is shown in green and α-syn is shown in red. Scale bar, 50 nm. F, G, The expression of α-syn in the media (F) and exosomes (G) from H4 cells. The total expression levels of α-syn in the media or exosomes were normalized by total protein amount (n = 3, *p < 0.01). H–K, The expression of α-syn in Triton-soluble fractions (H), SDS-soluble fractions (I), media (J), or exosomes (K) taken from WT and MUT fibroblasts; (n = 3, *p < 0.01). L–N, The expression of α-syn in Triton-soluble fractions (L), SDS-soluble fractions (M), and media (N) taken from PCNs where PARK9 expression was modulated (n = 3, *p < 0.01). Levels were normalized to total protein. The values represent mean ± SEM from three independent experiments. Data analysis was performed by one-way ANOVA and post hoc Tukey test (B, F–N).