Fig. 5: RNF181 associates with ERα and modulates ERα stability.

a Intracellular localization analysis of RNF181 and ERα by immunofluorescence assay. MCF7 cells were cultured in normal medium before fixation. Intracellular localization of RNF181 (green) and ERα (red) were shown. Nuclei (blue) were stained with 4',6-diamidino-2-phenylindole (DAPI). b Co-IP assay reveals association between endogenous RNF181 and ERαin MCF7 cells. MCF-7 cells were harvested with RIPA lysis buffer. CO-IP was performed using antibody as indicated. c In the presence of the proteasome inhibitor MG132, the stabilization effect of RNF181 on ERα did not further increase ERα protein levels. HEK293 cells were transfected with 2 µg RNF181 plasmid and 0.5 µg Myc-tag or Myc-RNF181 plasmids. After 24 h, cells were treated with 10 uM MG132/vehicle for 6 h. Cell lysates were prepared for Western blot analysis. The results are representative for three independent experiments. d RNF181 increases ERα half-life in HEK293 cells. HEK293 cells were transfected with HA-ERα plasmid and Myc-tag or Myc-ZNF213 plasmids. After 24 h, cells were treated with 100 µM cycloheximide/vehicle for indicated times. Cell lysates were prepared for Western blot analysis. The results are representative for three independent experiments. The ERα relative density was measured by Image J software.