Fig. 3. Prostate cancer-derived SPOP mutants fail to promote Geminin poly-ubiquitination.

a Schematic of domain organization of SPOP and SPOP mutations detected in 1013 cases of prostate cancers³⁰. b 293T cells transfected with Flag-Geminin in combination with empty vector (EV), SPOP WT, or mutants were harvested for co-IP and WB with indicated antibodies. c 293T cells were transfected with the indicated plasmids and harvested for IP to detect the ubiquitination of Geminin. d 293T cells were co-transfected with Flag-Geminin and SPOP WT or mutants and harvested for co-IP and WB. e 293T cells were co-transfected with Flag-Geminin and SPOP WT or mutants and harvested for co-IP and WB to detect Geminin poly-ubiquitination. f In vitro ubiquitination assay was performed by incubating the reconstituted E1 and E2 enzymes with CUL3/RBX1, Myc-SPOP WT, or mutants immunoprecipitated from 293T cells and GST-Geminin purified from E. coli. g, h Co-IP analysis of indicated proteins and ubiquitination in 293T cells transiently transfected with Flag-Geminin-WT or S202F mutant. Source data are provided in this paper or Mendeley database (10.17632/8n7xt5rkhc.1). Similar results for (b–h) panels were obtained in two independent experiments.