Fig. 10.

Developing Proximity Ligation Models to define AGR2-EpCAM localization in cells. A, Expression of AGR2 and its binding partner EpCAM in a panel of cancer cell lines. Breast cancer cell line (MCF-7) and esophageal cancer cell line (FLO-1, OE33, and OE19) was analyzed by Western blot using AGR2 polyclonal antibody K47 and EpCAM monoclonal antibody. Tumor suppressor protein p53 status was also analyzed p53 monoclonal antibody. β-actin was used as loading control. B, Representative image of a proximity ligation assay performed with antibody pair of AGR2 mouse monoclonal antibody and EpCAM rabbit polyclonal antibody (upper panel) or AGR2 rabbit polyclonal antibody and EpCAM mouse monoclonal antibody Ab (middle panel) in MCF-7 cells. PLA probes (Anti-rabbit PLUS probe and anti-mouse MINUS probe) were then added to the samples. Following ligation and amplification, protein-protein interaction complex was detected with green fluorescent probes (Duolink). Green fluorescence foci indicate the interaction between the two proteins. As a control, MCF-7 was not incubated with the antibody pair but incubated with proximity ligation assay probes (lower panel) that showed no or few foci. Scale bar 25 μm. C, As a negative control, proximity ligation assay also was also performed in cells that do not express AGR2 and EpCAM (FLO-1). AGR2 mouse monoclonal antibody and EpCAM rabbit polyclonal antibody was used to show that non-transfected FLO-1 demonstrated no significant amount of foci. Scale bar 10 μm. Nuclei were counterstained with DAPI and cells were visualized with an epifluorescence microscope.