Fig. 5: PSMD14 interacts with ERα AF1 ___domain through its UBD ___domain.

A Immunofluorescence staining assay showing the localization patterns of PSMD14 and ERα in MCF-7 cells. Intracellular localization of PSMD14 (green) and ERα (red) is shown. Nucleus (blue) were stained with DAPI. Scale bar, 20 µm. B, C Immunoprecipitation assay showing the endogenous interaction between PSMD14 and ERα. For examining the endogenous interaction between PSMD14 and ERα, lysates of MCF-7 cells were precipitated with anti-ERα or anti-PSMD14 antibodies, and the precipitates were examined by immunoblotting. D Schematic of the ERα protein, along with the ERα deletion mutants (residues 1–180, 1–300, 180–595 and 300–595) used in the Co-IP assays. E Schematic of the PSMD14 protein, along with the PSMD14 deletion mutants (residues 1–172 and Δ1–138) used in the Co-IP assays. F Immunoprecipitation assay showing PSMD14 interacts with ERα through its UBD ___domain (1–172) which contains MPN ___domain (58–138). HEK-293T cells were cotransfected with 2 µg ERα plasmid and full-length GFP-PSMD14 or its mutants (1–172, Δ1–138). After 24 h, the cells were treated with 10 μM MG132 for 6 h. Then, the cells were harvested with NP-40 lysis buffer. Co-IP was performed using an anti-Flag antibody, and the possible interacting PSMD14 domains were detected with anti-GFP antibody. G Immunoprecipitation assay showing AF1 ___domain is required for ERα to interact with PSMD14. HEK-293T cells were cotransfected with 2 µg PSMD14 plasmid and full-length HA- ERα or mutant ERα (1–180, 1–300, 180–595 and 300–595). After 24 h, the cells were treated with 10 μM MG132 for 6 h. Then, the cells were harvested with NP-40 lysis buffer. Co-IP was performed using an anti-Myc antibody, and the possible interacting ERα domains were detected with anti-HA antibody. H, I Immunoblot analysis showing the expression level of ERα protein in siControl and siPSMD14 expressing MCF-7 (H) and T47D (I) cells by treat with 10 μM proteasome inhibitor MG132. J–M Immunoblot analysis showing PSMD14 increased ERα half-life. siControl and siPSMD14 expressing in MCF-7 (J) and T47D (L). Cells were treated with 100 μM cycloheximide (CHX) for the indicated times. The expression of ERα protein was estimated by ImageJ software and is represented graphically in the right panel (K, M). N, O PSMD14 deubiquitinating enzyme activity deletion mutant cannot increase ERα half-life. HEK-293T cells were cotransfected with Flag-ERα plasmid and EGFP vector or EGFP-PSMD14 WT/mutants (H113Q/C120S/H113Q; C120S) plasmid for 24 h. Then cells were treated with 100 μM cycloheximide (CHX) for the indicated times. The expression of ERα protein was estimated by ImageJ software and is represented graphically in the right panel (O).