Figure 7. PINK1 accumulation following depolarization with CCCP may be required for Parkin recruitment.

(A) HeLa cells stably expressing YFP-Parkin were treated with 2 µM CCCP 1 h alone or CCCP 1 h + 2 µM CHX (30-min pretreatment and 1-h treatment) in the absence of serum. Whole-cell lysates were run on SDS gels and immunoblotted for endogenous PINK1 and the loading control GAPDH. (B) Cells treated as in (A) were fractionated. The mitochondria-enriched membrane fraction (Memb) was run on SDS gels and immunoblotted for endogenous PINK1 and VDAC. (C) HeLa cells were transfected with YFP-Parkin (green) and treated with 10 µM CCCP 1 h alone, CCCP + 10 µM of actinomycin (30-min pretreatment and 1-h treatment), or CCCP 1 h + 100 µM CHX (30-min pretreatment and 1-h treatment) in the presence of serum and immunostained for Tom20 (red). (D) Colocalization between YFP-Parkin and mitochondria in (C) was scored for ≥150 cells/condition in three or more independent experiments. (E) HeLa cells stably expressing YFP-Parkin were treated as in (A) and fractionated. The mitochondria-rich fraction was run on an SDS gel and immunostained for Parkin. Scale bars in all images represent 10 µm.