Fig. 1. DAPK1 binds to α-synuclein in human dopaminergic SH-SY5Y cells. (A) SH-SY5Y cells were transfected with plasmids encoding Myc-α-synuclein and/or FLAG-DAPK1 for 24 h. Total cell lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-Myc or anti-FLAG antibodies. Tubulin served as a loading control. (B) SH-SY5Y cell lysates were immunoprecipitated with anti-DAPK1 antibody, followed by immunoblotting with the indicated antibodies. Where indicated, cell lysates were immunoprecipitated with pre-immune IgG as a negative control. (C) Mouse primary cortical neurons were immunoprecipitated with anti-DAPK1 antibody, followed by immunoblotting with the indicated antibodies. Where specified, cell lysates were immunoprecipitated with pre-immune IgG as a control. (D) Representative confocal images of immunostaining of GFP-α-synuclein (green) and FLAG-DAPK1 (red) are shown. Scale bar=10 μm. Pearson’s correlation coefficient of the colocalization of α-synuclein and DAPK1 was calculated using Image J software. Data represent the mean±standard error of the mean (SEM) of five independent experiments (**p≤0.01). (E) Representative confocal images of immunostaining of endogenous α-synuclein (green) and DAPK1 (red) are shown. (F) Proximity ligation assays were performed either without antibody (control) or with the primary antibodies of α-synuclein and DAPK1.
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