Fig. 2: GLP is a ubiquitination and proteasomal degradation target of SPOP.

a Diagram showing the four GLP clones identified from Y2H screen using full-length SPOP as bait. Minimal interacting region shared by positive clones (the region between two dashed blue lines) covers two putative SBC motifs (red rectangles). b Western blots of co-IP samples from 22Rv1 cells treated with 20 µM MG132 for 8 h. c Representative images of proximity ligation assay (PLA) in 22Rv1 cells transfected with indicated plasmids. Scale bar, 10 μm. d Schematic representation of SPOP deletion mutants indicating their binding capability with GLP. e Western blots of WCL and co-IP samples from 293T cells transfected with indicated plasmids and treated with 20 μM MG132 for 8 h. f Western blots of WCL from 22Rv1 cells expressing indicated shRNAs. g RT-qPCR analysis of indicated genes in 22Rv1 cells expressing indicated shRNAs. Data shown means ± SD (n = 3 replicates/group). h Western blots of WCL from 293T cells transfected with the indicated plasmids. i, j Representative IFC images of Myc-SPOP and GLP staining in 22Rv1 cells transfected with Myc-SPOP WT (i). Scale bar, 10 μm. GLP staining was quantified using ImageJ optical density (OD)/nuclear area (pixel) (j). Data shown means ± SD (n = 50 cells/group). k Western blots of WCL from 293T cells transfected with indicated plasmids and treated with DMSO, MG132 (20 μM), bortezomib (200 nM) or chloroquine (50 μM) for 8 h. l Western blots of WCL and co-IP samples from 293T cells transfected with the indicated plasmids and treated with 20 µM MG132 for 8 h. m Western blots of the products of in vivo ubiquitination assays from 22Rv1 cells expressing indicated shRNAs and HA-Ub. n–o 22Rv1 cells expressing indicated shRNA were treated with 50 μg/ml cycloheximide (CHX) and harvested at different time points for western blots (n). Quantification of GLP protein from western blots normalized to actin and then to 0-h time point (o). Statistical significance was determined by unpaired two-tailed Student’s t test in (g, j). Experiments in (b, c, e, h, k, l) were repeated twice. Source data are provided as a Source Data file.