Figure 3. Pathogenic mutants of Parkin are subjected to Ser65 phosphorylation.

(a) Diagram of Parkin protein illustrating the pathogenic mutants used in this study. The Ser65 residue in the Ubl domain is shown as a yellow circle. RING, Ring-finger motif; IBR, in-between-Ring fingers domain. (b) Phos-tag Western blotting for Parkin and Western blotting for PINK1 were performed using Parkin WT and a series of pathogenic mutants as shown in Figure 2a. (c) Endogenous Parkin was also phosphorylated in SH-SY5Y cells after CCCP treatment. Post-nuclear cell lysates from SH-SY5Y cells treated with or without 10 µM CCCP for 30 and 60 min were fractionated into mitochondria-rich (Mito) and cytosolic (Cyto) fractions. These two fractions and their combination (Mito + Cyto) were subjected to Phos-tag or normal Western blotting analyses. Endogenous PINK1 was fractionated in the Mito fraction, as previously reported⁴⁵. Lactate dehydrogenase (LDH) and Tom20 were used as cytosolic and mitochondrial marker proteins, respectively. Asterisks: putative cleaved Parkin; dots: non-specific bands.