Fig 3.
Skp2 mediates ligand-independent proteasomal degradation of ERα with Cul7 and Rbx1 as part of the SCFSkp2 complex. (A) Ubiquitination assay of ERα in Cos-1 cells in the presence of vehicle or ligand (0.1% ethanol vehicle or 10 nM trans-hydroxy-tamoxifen [TOT]) and/or Skp2. (B) Ubiquitination assay of ERα in Cos-1 cells with pCMV-Skp2 or -ΔFbox-Skp2. (C) Ubiquitination assay for endogenous ERα in MCF-7 cells infected with the control or Skp2 adenovirus. (D) CoIP of Flag-Skp2 and various cullin isoforms in Cos-1 cells. (E) CoIP of Flag-ERα and various cullin isoforms in Cos-1 cells. (F) CoIP of Flag-ERα and HA-tagged pCMV-Rbx1 or -Rbx2 in Cos-1 cells. (G) Western analysis of MCF-7 cells transfected with siGL-3 or siRNA against various cullin isoforms or Rbx1, followed by ligand treatment. (H) Western analysis of MCF-7 cells transfected with siGL-3 or siRNA against various cullin isoforms or Rbx1, followed by infection with the control or Skp2 adenovirus. All data are representative of 3 experiments.