ERb Function in Mesothelioma Figure 4. ERb associates with EGFR and caveolin 1. A) Co-immunoprecipitation experiments were performed on REN cells treated 1 and 5 minutes with 5 ng/ml of human recombinant EGF. ERb and caveolin 1 were detected by Western blot in immunoprecipitations of membrane associated EGFR. B) Co-immunoprecipitation experiments were performed on mock and ERb over-expressing REN cells treated 5 minutes with 5 ng/ml of human recombinant EGF. ERb and caveolin 1 were detected by Western blot in immunoprecipitations of membrane associated EGFR. C) Confocal double fluorescent microscopy analysis of red-labeled ERb with green-labeled EGFR or caveolin 1 in mock- (left panel) or ERb-transfected (right panel) REN cells treated or not 5 minutes with 5 ng/ml of human recombinant EGF. D) Confocal fluorescent microscopy analysis showing the localization of green- labeled EGFR and phalloidin-TRITC labeled actin filaments in mock and in ERb and ERa transfected REN cells. Nuclei were counterstained with DAPI. doi:10.1371/journal.pone.0014110.g004 Cell cultures treatments and transfection The epithelioid MMe derived REN cell line that was used as the principal experimental model in this investigation was isolated, characterized [38] and kindly provided by Dr. S.M. Albelda (University of Pennsylvania, Philadelphia, PA) and the MSTO- 211H cell line established from the pleural effusion of a patient with biphasic mesothelioma of the lung [39] was obtained from the Istituto Scientifico Tumori (IST)-Cell-bank, Genoa, Italy. Cells were PLoS ONE | www.plosone.org 7 November 2010 | Volume 5 | Issue 11 | e14110