Figure 4.

Knock-down of endogenous PINK1 abrogates Parkin Ser⁶⁵ phosphorylation. (a) Timecourse of endogenous PINK1 stabilization by CCCP treatment. HEK293 cells were stimulated at the indicated time points with 10 μM of CCCP. One milligram of whole-cell lysates were immunoprecipitated with anti-PINK1 antibody (S085D) or pre-immune IgG covalently coupled to protein G Sepharose and resolved by 8% SDS-PAGE. Immunoblotting was performed with total PINK1 antibody (Novus). Representative of three independent experiments. (b) Knock-down of endogenous PINK1 abrogates Parkin Ser⁶⁵ phosphorylation. HEK293 cells were co-transfected with PINK1 siRNA (#1 or #2) or scrambled siRNA (scrambled) and untagged wild-type (WT) or Ser⁶⁵Ala (S65A) mutant Parkin as indicated using TransFectin reagent (Bio-Rad). Forty-eight hours post-transfection, cells were treated with or without 10 μM CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with GST-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser⁶⁵ antibody in the presence of dephosphorylated peptide. Five per cent of the IP was immunoblotted with total anti-Parkin antibody. 0.25 mg of whole-cell lysates were immunoprecipitated with anti-PINK1 antibody (S085D) and immunoblotted with anti-PINK1 antibody (Novus). Representative of three independent experiments.