HEK293 cells were either untransfected (lane one) or transfected with full length (1-581) PINK1-myc cDNA (lane two). 25μg of protein was subjected to SDS-PAGE analysis before being transferred to nitrocellulose membrane and immunoblotted with anti-PINK1 (186-202) at 1 μg/ml overnight at 4°C. Binding of the primary antibody was detected using rabbit peroxidase conjugated anti- sheep IgG antibody (1 in 2500 dilution) and enhanced chemiluminescence.

PINK1 was immunoprecipitated from 1mg HEK 293 cellular extracts over-expressing humanwildtypePINK1. Immunoprecipitateswereperformedusing5μgofpre-immune serum covalently coupled to protein G sepharose (lane 1)