Skip to main content
. 2021 Mar 10;11:638751. doi: 10.3389/fonc.2021.638751

Figure 5.

Figure 5

ZNF213 modulates ER alpha poly-ubiquitination and protein stability (A) In the presence of the proteasome inhibitor MG132, the stabilization effect of ZNF213 on ER alpha did not further increase ER alpha protein levels. HEK293 cells were transfected with 2 µg ER alpha plasmid and 0.5 µg Myc-tag or Myc-ZNF213 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. (B) ZNF213 increases ER alpha half-life in HEK293 cells. HEK293 cells were transfected with HA-ER alpha 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 ER alpha relative density was measured by Image J software. (C) ZNF213 increases ER alpha half-life in MCF-7 cells. Cells were transfected with siZNF213 or siControl. After 48 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. (D) The ZF domain is required for ZNF213 to stabilize ER alpha in HEK293 cells. (E) ZNF213 prohibits ER alpha poly-ubiquitination. HEK293 cells were transfected with 2 µg ER alpha plasmid and 0.5 µg Myc-tag or Myc-ZNF213 plasmids. After 24 h, cells were treated with 10 uM MG132 for 6 h. Cells were directly harvested and Western blot analysis using ER alpha antibody was used to detect ubiquitinated ER alpha forms. The predicted molecular weight of poly-ubiquitinated ER alpha is indicated. (F) ZNF213 decreases K48-linked poly-ubiquitination of ER alpha. HEK293 cells were transfected with 2 µg ER alpha plasmid, 0.5 µg HA-K48 Ubi plasmid, and 0.5 µg Myc-tag or Myc-ZNF213 plasmids. The cell extracts were immuno-precipitated with HA antibody. The K48 specific poly-ubiquitinated ER alpha was detected via western blotting analysis.