Fig. 3

SPOP/CUL3/RBX1 complex poly-ubiquitinates CYCLIN E1. a HEK293 cells were transfected with indicated plasmids for 24 h followed by MG132 (10 mM) treatment for 12 h. Immunoprecipitated Flag-CCNE1 were analyzed for ubiquitination with anti-HA WB. b SPOP/CUL3/RBX1 complex was expressed in insect cells and affinity purified. In vitro ubiquitination assay was performed together with bacteria-expressed E1, E2, and GST-CCNE1. c Flag-CCNE1 was expressed in HEK293 cells with SPOP wild-type or mutants. Twenty-four hours after transfection, cells were harvested for WB. d HEK293 cells were transfected with indicated plasmids. Co-immunoprecipitation was performed to study CYCLIN E1-SPOP interaction after treatment with 10 μM MG132 for 12 h. e HEK293 cells were transfected with indicated plasmids and treated with 10 μM MG132 for 12 h. Ubiquitination assay was performed to study the effect of SPOP mutants on CYCLIN E1 ubiquitination