Fig. 6: The 20S proteasome proteolytic signature in cells.

a Quantification of 20S proteasome levels in control and Dox-induced Hi20S cells. Data represents the average 20S proteasome content calculated from anti-PSMA1 IB of five independent experiments (±SD error bar). n = 5 sample points. *** represents p value < 0.0001 from unpaired t test. b Control and Hi20S cells transiently expressing either HA-tagged WT- or K0- Cyclin B1-NT were pulsed with 0.5 µM bortezomib for 6 h followed by cycloheximide chase. Residual Cyclin B1-NT was detected by IB. c Tables list all peptides of K0-Cyclin B1-NT identified from intracellular peptidomics of Hi20S and control cells, respectively. Peptides highlighted in light-blue match to peptides obtained from in vitro cleavage of unmodified Cyclin B1-NT by isolated 20S proteasomes (as identified in Fig. 2). The light-green highlighted peptides were identified in both cell types. Unique cleavage sites identified on K0-Cyclin B1-NT in Hi20S cells are marked by arrows (illustration below). The corresponding P1 positions match the preferred P1 positions for 20S proteasomes cleavage of unmodified Cyclin B1-NT identified in vitro (as in Fig. 2). d Size distribution of intracellular Cyclin B1-NT derived peptides obtained from control and Hi20S cells (from three data sets each cell line). n = 13, 50 sample points. e Size distribution of all intracellular peptides obtained in control or Hi20S cells from triplicate data sets. Boxplot on the right represents median peptide size. Box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. f Disorder score distribution of potential proteasome substrates unique to each cellular condition (control, Hi20S). Proteins were assigned from intracellular peptide captured and scored based on intrinsic disordered elements. Control n = 101 and Hi20S n = 787 over 3 independent experiments. * represents p value = 0.0215 from unpaired t test. g Control and Hi20S cells were pulsed with puromycin for 2 h. Residual puromycin-containing polypeptides were immunoprecipitated at 0 and 3 h of recovery, and IB for ubiquitin and puromycin content. h Control and Hi20S cells were pulsed with 0.5 µM bortezomib for 6 h and chased with cycloheximide for up to 6 h of recovery; samples taken for ubiquitin IB. For box plots a, d, and f, the center lines (with/without notch) show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by open circles; crosses represent sample means. Source data are provided as a Source data file.