FIGURE 8.

Human lung fibroblasts were growth-arrested and were then incubated in the presence (+) or absence (−) of TGFβ1 (10 ng/ml) for 72 h. A, the culture supernatant was collected and concentrated using a Micron centrifugal filter (Millipore) with a 3-kDa cut-off. The equivalent of 1 ml of supernatant was then loaded into each well of a 7.5% polyacrylamide gel. Western blotting was carried out to demonstrate the presence of the native proteins and higher molecular weight complexes of TSG-6, bikunin, and heavy chain 5. B, TGFβ1-treated cells were lysed and split into two identical samples of 30 μg. One half was treated with chondroitinase ABC (0.166 units/ml; Sigma-Aldrich), and both samples were incubated at 37 °C overnight. The samples were then separated on a 7.5% polyacrylamide gel, and Western blotting was carried out using anti-HC5 to demonstrate the degradation of the higher molecular weight complexes containing heavy chain 5. All blots in A and B are representative of three independent experiments.