Figure 4

CXCR7 co-localizes with EGFR. A-D. After fixation of MCF7 cells, PLA (in situ co-immunoprecipitation) was performed with specific CXCR7 [GTX100027, (1:500), GeneTex, Irvine, CA] and EGFR antibodies [E2760, (1:500), Sigma Aldrich, St. Louis, MO] to visualize heterodimerization of CXCR7 with EGFR. Colocalization is shown as a red fluorescent PLA signal (CX7/EGFR), and nuclei were counterstained with DAPI (blue) (630x magnification). As the PLA technique requires two specific antibodies to give a red fluorescent signal we used a single primary antibody as a negative signal control. A. Negative controls experiment using EGFR antibody alone. B. Basal level of CXCR7-EGFR colocalization in non-stimulated MCF7 cells. C. CXCR7-EGFR colocalization in MCF7 cells stimulated for 2 min with EGF (10 ng/mL). D. CXCR7-EGFR colocalization in EGFR over expressing (O.E.) MCF7 cells following 2 min EGF (10 ng/mL) stimulation. E. Immunofluorescence of normal human breast tissue for detection of CXCR7 (green fluorescence) and EGFR (red fluorescence) (400× magnification). F. Immunofluorescence of ER + breast cancer tissue stained as explained in E. G. PLA of normal human breast tissue using CXCR7 and EGFR antibodies to visualize colocalization of CXCR7 with EGFR (CX7/EGFR), nuclei were counterstained with DAPI; 400× magnification. H. PLA of ER + human breast tumor tissue stained as explained in G.