Figure 5
From: The chemokine receptor CXCR7 interacts with EGFR to promote breast cancer cell proliferation
β-arrestin2 plays a role in CXCR7 mediated phosphorylation of EGFR and ERK. A. Levels of pERK1/2 and total ERK in MCF7 cells transfected with siCXR7 or control siRNA (C). ERK levels were determined by Western blotting of cell lysates prepared from serum-starved cells stimulated with EGF (10 ng/mL) for 5 minutes. B. The ratio of p-ERK1/2 over total ERK1/2 in western blot was quantified from analysis of MCF7 cell lysates that were transfected with control plasmid (cDNA), β-arrestin2 (βAR2) or β-arrestin2 in combination with siCXR7 (βAR2siR7) and stimulated with EGF (+). C. CXCR7 down regulation via siRNA decreased activation of EGFR at Tyrosine 1110 in MCF7 cells. D. The ratio of p-EGFRY1110 over total EGFR was quantified in cells transfected with control plasmid (cDNA), β-arrestin2 (βAR2), or β-arrestin2 in combination with siCXR7 (βAR2siR7) and stimulated with EGF (+). The western blot experiments were repeated twice and one representative experiment is shown. E-H. Following transfection with EGFR-WT plasmid for EGFR over expression (O.E.), PLA was performed in MCF7 cells to visualize heterodimerization of CXCR7 with EGFR after 5 min EGF (10 ng/mL) stimulation; nuclei were counterstained with DAPI (blue). E Incubation with non-specific isotype antibody (negative control). F. PLA signal indicating CXCR7/EGFR dimers in non-stimulated MCF-7 cells. G. PLA signal indicating CXCR7/EGFR dimers in MCF7 cells transfected with EGFR-WT plasmid and siRNA against βarrestin2. H. CXCR7/EGFR dimers in MCF7 cells transfected with EGFR-WT plasmid and siRNA against CXCR7 (630x).