Fig. 2. DAPK1 directly phosphorylates α-synuclein at Ser129. (A) SH-SY5Y cells were either mock-transfected or transfected for 24 h with plasmid encoding FLAG-tagged wild-type DAPK1 (DAPK1-WT) or its kinase-defective mutant having the point mutation K42A (DAPK1-K42A). Cell lysates were immunoblotted with the indicated antibodies. (B) DAPK1-WT mouse embryo fibroblasts (MEF) or DAPK1-KO MEFs were treated either DMSO (control) or 100 nM rotenone for 24 h. Cell lysates were immunoblotted with the indicated antibodies. (C) SH-SY5Y cells were transfected with plasmids encoding FLAG-DAPK1 and/or various point-mutants of Myc-α-synuclein. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with the indicated antibodies. All graphed data represent the mean±SEM of five independent experiments (**p≤0.01; a-c). Statistical analyses were performed using the SPSS Statistics software (version 23.0) (IBM). (D, E) After SH-SY5Y cells were transfected for 24 h with plasmids encoding FLAG-tagged DAPK1-WT or DAPK1-K42A mutants, in vitro kinase assay of cell lysates was performed.
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