Fig. 4: GK13S, but not GK16S, reduces monoubiquitin in U-87 MG cells and thereby phenocopies the effect of a UCHL1 mutant mouse.

a Representative microscopy images of U-87 MG cells were taken 72 h after treatment with 1.25 µM of the indicated compounds (including the control Staurosporine, STS). Effects of the compounds on cell growth and viability were assessed using analysis of confluency (bright-field, BF) and apoptosis (propidium iodide staining, PI). See Supplementary Fig. 7a–d for quantitation. b Cellular activity-based protein profiling of intact U-87 MG cells treated with indicated compounds or DMSO for 1 or 24 h. c Schematic representation of overlapping and individual cellular targets of GK13S and GK16S, supporting their application as chemogenomic probes for the investigation of UCHL1. Compare Fig. 2e. d Inhibition of cellular UCHL1. Western blot analysis of endogenous UCHL1 labeled with HA-Ub-VS after treatment of U-87 MG cells with either the indicated compounds or DMSO for 1 or 24 h. e Western blots showing monoubiquitin levels in U-87 MG cells treated with GK13S or GK16S. An additional knockdown of UCHL3 does not aggravate the effect. f Quantification of monoubiquitin intensities upon treatment of U-87 MG cells with DMSO, GK13S, or GK16S (as shown in e). Values correspond to the mean of four or six independent experiments for control or UCHL3 knockdown, respectively. Error values represent the standard error of the mean (s.e.m.). Statistical significance was analyzed using individual one-sample, two-tailed t-tests compared to the mean of “1” as set for the DMSO-treated samples. **p < 0.01 (exact p = 0.0095 for DMSO/GK13S comparison in siScr background and p = 0.0014 for the DMSO/GK13S comparison in the siUCHL3 background); ns, not significant. Uncropped versions of gels and blots are shown in the supplementary information. Source data are provided as a Source Data file.