Figure 4

Interleukin-like epithelial-to-mesenchymal transition inducer binds to soluble and deposited fibronectin-containing extracellular matrix complexes via its propeptide. (A) Western blot analysis of wild-type (wt) or cleavage mutant (FD) interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) immunoprecipitated (IP) from conditioned medium of overexpressing EpRas cells via their FLAG epitope tags. Coprecipitation of extracellular soluble fibronectin was probed using a fibronectin-specific antibody. (B) Immunofluorescence analysis of purified WT and cleavage mutant (FD) ILEI-6xHis proteins after incubation with prepared cell-free extracellular matrix (ECM) deposited by NIH 3T3 fibroblasts. Bound ILEI proteins were visualized via the 6xHis affinity tag using penta-His antibody (green). ECM was counterstained with a fibronectin-specific antibody (red). The purification fraction of empty vector expressing EpRas cells was used as control. Scale bar, 5 μm.