Fig. 4

SMURF1 associates with ER alpha and increases its stability. a Co-IP assay reveals association between endogenous SMURF1 and ERα in MCF7 cells. MCF-7 cells were harvested with NP-40 lysis buffer. CO-IP was performed using antibody as indicated. b SMURF1 is mainly localized in the cytoplasm and associates with ER alpha in the cytosol. The subcellular protein fractionation kit (Thermo scientific, 78,840) was used for cytoplasm and nuclear separation. Tubulin and Histone-3 were used for cytoplasm and nuclear control. Based on the separation, IP was done by SMURF1 antibody in both the cytosol and nuclear lysis. ER alpha antibody was used to detect the interaction in both the cytosol and nuclear. c Intracellular localization analysis of SMURF1 and ER alpha by immunofluorescence assay. MCF7 cells were cultured in phenol red-free DMEM medium. Intracellular localization of SMURF1 (red) and ER alpha (green) were shown. Nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI). d In the presence of the proteasome inhibitor MG132, the stabilization effect of SMURF1 on ER alpha did not further increase ER alpha protein levels. HEK293 cells were transfected with 2 μg ERα plasmid and 0.5 μg Myc-tag or Myc-SMURF1 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. e and f SMURF1 increases ERα half-life in HEK293 cells. HEK293 cells were transfected with HA-ERα plasmid and Myc tag or Myc-SMURF 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 ER alpha relative density was measured by Image J software. g SMURF1 prohibits ERα poly-ubiquitination. HEK293 cells were transfected with 2 μg ERα plasmid and 0.5 μg Myc-tag or Myc-SMURF1 plasmids. After 24 h, cells were treated with 10 uM MG132 or vehicle for 6 h. Cells were directly harvested and Western blot analysis using ERα antibody was used to detect ubiquitinated ERα forms. The predicted molecular weight of polyubiquitinated ERα is indicated