Detection of Phospho-Ubiquitin (S65) by Western blot.Tetraubiquitin chains of each indicated linkage type were incubated for 0-4 hours in reactions containing recombinant PINK1 kinase (Cat# AP-180) and ATP. At indicated times a portion of each reaction was removed and terminated with SDS-PAGE sample buffer. SDS-PAGE gels (10-20%) were used to resolve approximately 150 ng of Ubiquitin tetramer from each reaction. Western Blots were developed using eitheralpha -phospho-Ubiquitin, pS65 (upper panels) or anti-Ubiquitin (Cat# MAB701, lower panel). Primary antibodies were used at 1 μg/ml in PBST + 0.5% BSA, while HRP-labeled secondary antibodies ( alpha -rabbit for A-110,alpha -mouse for MAB701) were used at a 1:10,000 dilution in PBST + 0.5% BSA. Further details are available upon request.

Detection of Phospho-Ubiquitin (S65) by Western blot.Tetraubiquitin chains of each indicated linkage type were incubated for 0-4 hours in reactions containing recombinant PINK1 kinase (Cat# AP-180) and ATP. At indicated times a portion of each reaction was removed and terminated with SDS-PAGE sample buffer. SDS-PAGE gels (10-20%) were used to resolve approximately 150 ng of Ubiquitin tetramer from each reaction. Western Blots were developed using eitheralpha -phospho-Ubiquitin, pS65 (upper panels) or anti-Ubiquitin (Cat# MAB701, lower panel). Primary antibodies were used at 1 μg/ml in PBST + 0.5% BSA, while HRP-labeled secondary antibodies ( alpha -rabbit for A-110,alpha -mouse for MAB701) were used at a 1:10,000 dilution in PBST + 0.5% BSA. Further details are available upon request.