Complex formation between PEDF and component(s) in media conditioned by human retinoblastoma Y-79 cells. Binding reactions were performed with 0.75 nM 125I-PEDF and human retinoblastoma Y-79 cell conditioned media (CM) (200 μl) at 4°C for 120 min with gentle rotation. Free and bound PEDF was separated by ultrafiltration through membrane of MWCO = 100 kDa. PEDF bound was calculated by subtracting specific 125I-PEDF binding in fresh media from that in CM and is given as a ratio of the total 125I-PEDF in the reaction. A) Reactions with CM (CM 1X) and 10-fold concentrated CM (CM 10X). B) Reactions with protease-treated CM. CM was concentrated 10-fold and dialyzed against 20 mM Tris pH 8, 10% glycerol. Half of the dialyzate was incubated without and the other with 0.4 μg/ml subtilisin at 37°C for 16 h (-Subt. and +Subt., respectively). The protease reaction was terminated by incubation at 75°C for 25 min to inactivate the protease. All experimental points are given as the average of duplicates. Assays were performed twice.