FIGURE 7.

A, the absolute copy number of each IαI heavy chain was determined by reading standard curves prepared by adding known numbers of molecules to the qPCR. Heavy chain 5 was shown to be the predominant isoform. Results are mean ± S.E. of four independent experiments. B, human lung fibroblasts were growth-arrested and were then cultured in the presence or absence of TGFβ1 (10 ng/ml) for 72 h. The cells were lysed with radioimmune precipitation assay buffer, and 30 μg of protein was separated on 7.5% polyacrylamide gels. GAPDH antibody was used as a loading control. Blots shown are representative of four experiments. Top, Western blot showing that the amount of IαI heavy chain 5 present was markedly increased following stimulation with TGFβ1 Bottom, following TGFβ1 incubation, the cells were treated with 10 μg/ml trypsin in PBS for 10 min at room temperature as shown previously to remove any cell surface/CD44-associated HA (43). C, cells were incubated with or without TGFβ1 (10 ng/ml) for 72 h. The cells were then either left untreated or exposed to Streptomyces hyaluronidase (0.4 units in 400 μl of PBS for 2 h). All cells were then fixed with 4% paraformaldehyde in PBS for 20 min and stained with anti-HC5 antibody followed by FITC-conjugated anti-rabbit IgG to demonstrate the presence of heavy chain 5 on the cell surface. Data shown are representative of three independent experiments.