ERβ over-expression influences EGFR mediated signaling and internalization.

<p><b>A</b>) The graph show the growth curves of mock- and ERβ-transfected REN cell treated for 24 and 48 hours with 5 ng/ml of EGF in 2% FBS culture medium. At each time point, the cells were assayed for proliferation. Each value represents mean ± SD (n = 3). Adjacent to the graph is reported a representative Western blot analysis that documents ERβ expression. Tubulin staining indicates equal loading of the proteins. <b>B</b>) Mock- and ERβ- transfected REN cells made quiescent for 2 hours were treated with 5 ng/ml of EGF for 5 minutes and detergent extracted. Levels of phosphorylated EGFR, ERK 1/2 MAP kinases and Akt were analyzed by immunoblotting. Membranes were also blotted with antibodies to EGFR, Erk1/2 and Akt to evaluate protein expression. Tubulin was blotted to show equal amount of loading. Western blot analysis with anti ERβ antibodies documents its expression in transfected cells. Representative of three separate experiments. <b>C</b>) Evaluation of EGFR internalization was performed by Flow cytometry analysis on wild type and ERβ expressing REN cells treated 60 or 120 minutes with 10 ng/ml of human recombinant EGF. Histograms represent percentage of positive cells following incubation with anti-EGFR antibody indicated for each condition ± SD. Data are representative of three separate experiments. <b>D</b>) Representative immunoprecipitation experiment of membrane associated EGFR performed on mock and ERβ over-expressing REN cells, treated 60 or 120 minutes with 10 ng/ml of human recombinant EGF. Membrane was blotted with anti-pY and anti-EGFR antibodies.