Figure 4
Interaction of endogenous ERβ with EGFR. A and B: H1793, A549, H1944 and H1792 cells were treated with 10 nM E2, 10 ng/ml EGF, or the combination for 1 h. 300 μg WCE were incubated with rabbit polyclonal ERβ antibody (Millipore) and them immunoprecipitate using a Protein G beads (Cell Signaling) or rabbit IgG control, and immunobloted using ab EGFR (1005). A: EGFR bands were identified in the IP of cytoplasmic (CE) and nuclear (NE) extracts of H1793 cells treated with EtOH and E2, but not EGF or the combination of E2 and EGF treated H1793 cell. EGFR bands were identified in the IP of cytoplasmic (CE) extracts of A549 cells treated with EGF or the combination of E2, but not EtOH and E2 treated cells. B: EGFR was identified in the ERβ IP from H1944 cells treated with EtOH and E2, but not EGF or the combination of E2 and EGF. EGFR bands were not identified in the IP from male derived H1792 cells. The membranes were stripped and reprobed for ERβ. C: The subcellular localization of EGFR and ERβ in lung adenocarcinoma cells was examined by immunofluorescent staining. The merged images are shown with anti-mouse EGFR Ab-13 (green) and anti-rabbit ERβ ab (06-629) (red) and counterstaining with DAPI (blue) in EtOH- and E2, EGF, E2+EGF treated H1793, H1792, H1944 and A549 cell lines. The individual images for each staining in Additional figure 11, Figure S7.