Analysis of Miro1 ubiquitin chains.
A, ubiquitination assays performed in SH-SY5Y cells stably overexpressing FLAGParkin and transfected with either wild type HAubiquitin or several ubiquitin mutants that allow only specific chain types (Lys-6, Lys-11, Lys-27, Lys-29, Lys-33, Lys-48, and Lys-63) reveal that Miro1/2 is primarily ubiquitinated in a Lys-27-dependent manner; n = 3. B, same assay is carried out using HAubiquitin mutants that cannot mediate specific ubiquitin chain linkages (K6R, K11R, K27R, K27R/K29R, K48R; n = 3). C, immunopurified GFPMiro1 was incubated with increasing amounts of OTUD2 (that specifically cleaves Lys-11, Lys-27, Lys-29, and Lys-33, mediated ubiquitin linkages), Otulin (cleaves Met-linked chains) or USP21 (chain type aspecific DUB). Black arrowheads indicate HA-positive bands, likely to be ubiquitin trimers and dimers; asterisk indicates cross-reactivity between Otulin and the anti-HA antibody. IP, immunoprecipitation.