Fig. 6

USP36 associates with ERα and modulates ERα stability in breast cancer cell. A Immunofluorescence staining assay showing the localization patterns of USP36 and ERα in MCF-7 cells. Intracellular localization of USP36 (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 USP36 and ERα. For examining the endogenous interaction between USP36 and ERα, lysates of MCF-7 cells were precipitated with anti-ERα or anti-USP36 antibodies, and the precipitates were examined by immunoblotting with 2% input sample. 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. Schematic of the USP36 protein, along with the USP36 deletion mutants (residues 1–420; 421–800 and 801–1121) used in the Co-IP assays. E–F Immunoprecipitation assay showing AF1 domain is required for ERα to interact with USP36. HEK293T cells were co-transfected with 2 µg USP36 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-Flag antibody, and the possible interacting ERα domains were detected with anti-HA antibody. G Immunoprecipitation assay showing USP36 interacts with ERα through its USP domain (1–420). Co-IP was performed using an anti-HA antibody, and the possible interacting USP36 domains were detected with anti-Flag antibody. H; K; N USP36 and ERα protein levels were determined by western blotting. Cells by treat with 10 μM proteasome inhibitor MG132 for 6 h. I-J; L-M; O-P USP36 and ERα protein levels were determined by western blotting. MCF-7 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 (J, M, P). In Panels A-C, E-I, K-L, N–O, the results are representative of three independent experiments. All the data are presented as the means ± SDs. *P < 0.05; **P < 0.01 (Student’s t test)