Fig. 5. The neutral PRL-cleaving proteases are secreted by chondrocytes and identified as MMPs. (A) Reducing western blot analysis of proteolytic products generated from PRL by incubation of 200 ng of human PRL with 5 µl of conditioned medium (CM) or non-conditioned medium (NCM), obtained after incubation with or without rat chondrocytes, respectively. Arrows indicate the 16 kDa fragment. (B) Reducing western blot analysis of proteolytic products generated from PRL by incubation of human PRL with non-conditioned medium (lane 1) or with chondrocyte-conditioned medium either alone (lane 2) or together with the serine protease inhibitor aprotinin (10 µg/ml), cysteine protease inhibitor E-64 (25 µg/ml), aspartyl protease inhibitor pepstatin A (Pepst A, 1.4 µM), MMP inhibitors EDTA (5 mM) and 1,10-phenanthroline (Phenant, 10 mM), or after heat inactivation (90°C for 30 min) of conditioned medium (). (C) Reducing western blot analysis of proteolytic products generated from PRL by incubation of human PRL with chondrocyte conditioned medium in the absence or presence of the MMP inhibitor GM6001 (10 µg/ml). (D,E) Reducing (R) and non-reducing (NR) western blot analysis of proteolytic products generated from PRL by incubation of 8 µg of human PRL with or without 10⁶ chondrocytes in the absence or presence of the MMP inhibitor GM6001 (10 µg/ml). Arrows indicate 16 and 25 kDa fragments. Positions of the PRL isoforms of the indicated relative molecular mass are shown on the left of western blots.