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. Author manuscript; available in PMC: 2022 Nov 17.
Published in final edited form as: Mol Pharm. 2021 Feb 18;18(3):1455–1469. doi: 10.1021/acs.molpharmaceut.1c00018

Figure 4. E2-PROTACs selectively reduce the expression of native and recombinant GPER.

Figure 4.

(A) HEK-293 cells stably expressing HA-GPER, HA-β1AR, or HA-CXCR4 were treated with 100 μM of UI-EP001, UI-EP002, and partial PROTAC for 1 hour. Fixed cells were permeabilized and then labeled with rabbit HA antibody and total receptors were then visualized using Alexa Fluor 594 anti-rabbit secondary antibody (red). (B) Corrected Total Red Fluorescence (CTRF) from images of HA-GPER, HA-β1AR, and HA-CXCR4 cells treated with either vehicle, UI-EP001, UI-EP002, and partial PROTAC was measured using Image J software from three different microscopic fields (***, P <0.0004; ****, P <0.0001; one-way ANOVA). (C) MCF-7 (ERα+, ERβ+, PR+, GPER+) cells were incubated at 37°C with vehicle, UI-EP001, UI-EP002, or partial PROTAC for 1 hour and then immunostained with mouse ERα, rabbit ERβ, mouse PR, and rabbit GPER antibodies and detected with either Alexa 488-conjugated anti-mouse IgG or Alexa 488-conjugated anti-rabbit IgG (green). (D) Quantification of results from images in (C) measured as CTRF. (E) SKBR3 (ERα+, ERβ−, GPER+) cells were incubated with vehicle (1% DMSO), 100 uM of UI-EP001, UI-EP002, or partial PROTAC for 1 hour. Surface and intracellular GPER were visualized in intact or detergent permeabilized cells, respectively using rabbit GPER antibodies and Alexa 594-conjugated goat anti-rabbit IgG (red).