Fig. 2. Identification of Ub sites and substrates by mass spectrometry.
a Identified His10-Ub substrates purified from cells treated for 3 h with indicated UPS inhibitors after removing proteins identified in parental U2OS samples were filtered out (all treatments) (right-sided student’s t test p value=0.05, S0 = 0.1), n = 3 biologically independent samples. Each data point is represented by a coloured circle; the box contains the 25–75th percentile and the orange line denotes the mean and the error bars represents the SD. b Identified Ub sites purified with UbiSite antibody from U2OS cells treated with the indicated UPS inhibitors for 3 h, n = 3 biologically independent samples. Each data point is represented by a coloured circle, the box contains the 25–75th percentile and the orange line denotes the mean and the error bars represents the SD. c Percentage of proteins in His10-Ub samples that changed significantly in any of the UPS inhibitor treatments and timepoints in comparison to DMSO control (student’s t test FDR = 0.05, S0 = 0.1). d Percentage of Ub sites that changed significantly in any of the UPS inhibitor treatments in comparison to DMSO control (student’s t test FDR = 0.05, S0 = 0.1). e, f Percentage of significantly altered proteins in response to the indicated treatment at the indicated timepoints in His10-Ub samples (e) and Ub sites at 3 h (f). g Venn diagram of Ub sites identified per treatment from UbiSite DDA data, where sites identified in at least one replicate in multiple treatments were considered as intersections. h Histogram of number of Ub sites identified per protein. i Scatter plot of number of Ub sites compared to the molecular weight of the protein, with Pearson correlation analysis. j UPS inhibitor treatments, and combinations of UPS inhibitors with or without addition of 50 µg/ml translation inhibitor Cycloheximide (CHX) at 3 h; samples were immunoblotted for Ub (P4D1), n = 3 biologically independent samples. Source data are provided as a Source Data file.