Effect of inhibitors on in vitro processing. Various concentrations of protease inhibitors were added to the in vitro processing assay for 6 hr at 29°C. The first lane is P25K expressed alone with no extract added. The second lane is P25K mixed with cellular extracts and the third lane is P25K mixed with I7L enzyme extracts. Each of the remaining lanes has P25K mixed with I7L enzyme extracts plus indicated inhibitor. Ethylenediaminetetraacetic acid (EDTA) was used at 1 mM. Pepstatin A, Pep, was used at 10 μM. Phenlymethanesulfonyl fluoride (PMSF) was used at 1 mM. N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) and a related product EST, were both used at 10 μM and 100 μM concentrations. Iodoacetic acid (IA) was used at 1 mM. Leupeptin (Leu) was used at 1 mM, and N-ethlymaleimide (NEM) was used at 2.5 mM. The concentrations of TTP-6171, TTP-1021, and TTP-0961 are indicated. The table indicates the concentration of inhibitor used and whether cleavage activity was observed.