HEK293 cells were either transfected with GFP empty vector/ GFP-PINK1 (125-end) mouse/ GFP-PINK1 (125-end) human and 25μg of protein was subjected to SDS-PAGE analysis before being transferred to nitrocellulose membrane and immunoblotted with anti-PINK1 (235-end) 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 mouse 25 GFP-PINK1 (125-END). Immunoprecipitation were performed using 5ug of pre-immune serum covalently coupled to protein G sepharose (lane1) or 5ug anti-PINK1 S774C antibody covalently coupled to protein G sepharose (lane 2). Immunoprecipitates were subjected to SDS-PAGE analysis before being transferred to nitrocellulose membrane and immunoblotted with anti-PINK1 IB: HAX (DSTT A IB: OMI (DSTT Ab) S774C antibody 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.