Generation and Characterization of Parkin Antibodies

Rabbit anti-Drosophila Parkin polyclonal antibody was raised against recombinant MBP-tagged Drosophila Parkin (275–482 aa) produced in the E. coli strain Rosetta 2 (Novagen). The antibodies used in the western blot analysis were as follows: anti-Parkin (1∶5,000 dilution), anti-Mfn (1∶2,000 dilution; a kind gift of Dr A. Whitworth), anti-Miro (1∶2,000 dilution; a kind gift of Dr E. Zinsmaier), anti-Drp1 (1∶2,000 dilution; a kind gift of Dr L. Pallanck), anti-ATP5A (1∶10,000 dilution; Abcam, 15H4C4), anti-NDUFS3 (1∶10,000 dilution; Abcam, 17D95), anti-Actin (1∶10,000 dilution; Millipore, MAb1501) and anti-Hsp60 (1∶1,000 dilution; Cell Signaling, D307). The antibody used in immunocytochemistry was anti-TH (1∶1,000 dilution; ref. [11] (link)). Complementary DNAs for human and Drosophila Parkin and PINK1 were described in previous studies [11] (link), [14] (link), [45] (link). Parkin phospho-mutants were generated by PCR-based mutagenesis followed by sequence confirmation of the entire gene.