Consistent with our biochemical and structural data and the proposed model for USP53 activity, we found elevated ubiquitination on the two core components of tricellular junctions MARVELD2 and LSR upon knockdown of USP53. The combined use of diglycine proteomics, different ubiquitin enrichment techniques and UbiCRest-like assays validates these as USP53 substrates and synergizes with recent advances in the assessment of ubiquitin chain architecture3,7,65 These substrates are supported by coinciding phenotypes of deafness and cholestasis36,47, consistent with the USP53 mouse phenotype30,47. The gene discussed is LSR; the disease is deafness.