In vitro kinase assay.

In vitro kinase assay was performed with purified recombinant human c-Abl and α-synuclein. Proteins were incubated in a kinase assay buffer (20 mM HEPES, pH 7.5, 5 mM EGTA, 20 mM β-glycerophosphate, 20 mM MgCl₂, 10 μM ATP, and 0.5 μCi of γ-³²P-ATP at a combined volume of 30 μl). The reaction mixture was incubated at 30°C for 30 minutes. Reactions were quenched by the addition of SDS–sample loading buffer, heated to 70°C for 10 minutes, and then loaded onto 4%–20% polyacrylamide gels for SDS-PAGE. Following electrophoresis, gel was transferred to PVDF membrane, heat-sealed in hybridization bags, and exposed to a phosphoimaging screen overnight at room temperature for assessing radioactive ³²P incorporation.