Interleukin-like epithelial-to-mesenchymal transition inducer is processed extracellularly by serine proteases, most efficiently by plasmin. (A) Western blot analysis of interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) in whole-cell lysate and conditioned medium (CM) of EpRas cells grown in 10% fetal calf serum (FCS) or under serum-free conditions. Loading was not normalized to cell numbers. Full-length ILEI runs at the size of 26 kDa, the processed form approximately 2 kDA lower. (B) Purified full-length ILEI-6xHis was incubated with serum-free Dulbecco’s modified Eagle’s medium (DMEM) or medium containing 10% FCS (DMEM 10%) for 5 hours. ILEI protein was repurified via its 6xHis affinity tag and subjected to Western blot analysis. (C) Western blot analysis of ILEI in CM of EpRas cells cultured in the presence of the broad-specificity inhibitors against cysteine proteases (E64), cathepsin B (CA-074), matrix metalloproteases (GM6001) and serine-type proteases (4-(2-aminoethyl)-benzolsulfonylfluorid-hydrochloride (AEBSF) and aprotinin) for 24 hours. (D) to (F) Purified intracellular, full-length ILEI-6xHis was subjected to Western blot analysis after incubation for 2 to 5 hours with the indicated amounts of purified (D) plasmin, uPA and tPA; (E) plasmin, thrombin and plasma kallikrein; and (F) plasmin, thrombin and neutrophil elastase (NE).