A HeLa cell line expressing PINK1 fused to GFP from the endogenous locus was generated using CRISPR–Cas9 nickase–mediated genome editing with homology-directed repair. GFP-positive clones were isolated by fluorescence-activated cell sorting.
Ubiquitinated proteins were enriched using Halo-m-DSK ubiquitin capture, and GFP-PINK1 complexes were isolated by IP, followed by SDS–PAGE and immunoblotting to assess PINK1 activation and downstream ubiquitination signaling upon GTPP treatment
