Interleukin-like epithelial-to-mesenchymal transition inducer secretion levels correlate with altered subcellular localization of the protein. (A) Western blot analysis of interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) in whole-cell lysates and conditioned medium (CM) of wild-type ILEI-overexpressing EpC40 cells (EpC40-wt) harvested 32 hours after serum withdrawal with or without treatment with plasmin (10 mU/ml) for 8 hours before harvest or aprotinin (10 μM) for 32 hours before harvest. CM of the last 24 hours were collected. (B) to (D) Immunofluorescence analysis of EpC40-wt cells as described in (A) without treatment (B) or after plasmin treatment (C) or aprotinin treatment (D). ILEI is visualized via its FLAG tag (green), Golgi is marked by a GM130 antibody (red) and genomic DNA is counterstained with DAPI (4′,6-diamidino-2-phenylindole, blue). Scale bar, 10 μm. (E) to (L) Immunohistochemistry for ILEI localization on representative 3-μm-thick sections of paraffin-embedded EpC40 mammary tumors (n = 5 per group) overexpressing the empty vector control (cont.; (E) and (F)) or the following ILEI constructs: EpC40-wt (G) and (H), EpC40-ΔN-RS (I) and (J) and EpC40-FD (K) and (L). (E, G, I and K) Tumors from nontreated animals. (F, H, J and L) tumors from aprotinin-treated animals. Overexpressed ILEI is shown via its FLAG epitope tag (brown), and genomic DNA is counterstained with hematoxylin (blue). Scale bar, 10 μm.