Research Paper Volume 14, Issue 3 pp 1200—1213

Figure 3. CCN1 directly binds the estrogen receptor and regulates its transcriptional activity. (A) MCF-7/pBABE, MCF-7/CCN1, MCF-7/D125A-CCN1, and MCF-7/TM-CCN1 cells were transiently with an ERE-Luciferase reporter (the ERE-containing reporter plasmid) and pRL/CMV (an internal reporter plasmid to control for transfection efficiency). Cells were incubated in the absence or presence of estradiol (E_2, 10⁻⁹ M), tamoxifen (10⁻⁷ M), fulvestrant (10⁻⁷ M), their combinations, or vehicles for 24 h, and cell extracts were analyzed for Luciferase activity. Data shown represent mean (columns) ± S.D. (bars) (n=3). (B) Top: Microphotographs show representative in situ immunofluorescence staining of CCN1 and/or estrogen receptor (ERα) in MCF-7/CCN1 cells. Scale bar is 10 μm. Bottom: ERα in the cell lysates of MCF-7 and MCF-7/CCN1 cells was immunoprecipitated and immunoblotted with anti-ERα and anti-CCN1 antibodies. (C) Representative immunoprecipitation results of His-tagged ERα and GST-CCN1 using immobilized Ni²⁺. Purified GST-CCN1 protein was incubated with human recombinant ERα-His protein and Ni-NTA His•Bind resin beads. As controls, ERα-His protein was incubated with GST-only beads or GST-CCN1 was incubated with Ni-NTA beads alone. Proteins retained in the beads were denatured and blotted with the indicated antibodies. Results in (B, C) are representative of three independent experiments. (S: Short exposure; L: Long exposure).