Extended Data Fig. 2: Cholestasis-associated patient mutations of USP53 are catalytically impaired.
From: Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53
a. Ub-RhoG cleavage assay. Indicated concentrations of USP5320-383 (WT or patient mutants) were added to Ub-RhoG and fluorescence was recorded over time. Data are shown as average of technical triplicates. b. Ub-RhoG cleavage assay with USP5421-385, shown as in panel a. c-d. Protein stability assessment of USP5320-383 (panel c) or USP5421-369 (panel d) proteins by thermal shift analysis. Averaged fluorescence raw data are shown and melting temperatures (Tm) are plotted from technical triplicates (USP53) or quadruplicates (USP54). e-f. Probe reactivity assay. HA-Ub-VS was incubated with indicated USP5320-383 (panel e) or USP5421-369 (panel f) proteins. Probe reactivity was analyzed by SDS-PAGE and Coomassie staining. Uncropped versions of all gels are provided as Source Data. g. Schematic of the HA-Ub-VS probe reactivity assay.
