Figure 5 shows the cell-growth assa

Figure 5 shows the cell-growth assay results for NIH 3T3 cells in the presence of soluble IGF-Y, commercial IGF-1 (c-IGF), and bound IGF-X. The addition of soluble IGF-1 to the cells led to a slight decrease in cell growth activity compared with the untreated cells. However, the treatment of these proteins with tyrosinase led to a sig- nificant increase in their cell growth activity. For example, a very small amount of bound IGF-X led to similar increases in the cell- growth activity as for soluble c-IGF. Nota- bly, the treatment of the cells with IGF-X led to a much greater increase in cell-growth activity than for soluble c-IGF. Similar results to these have also been observed for other immobilized growth factors and attributed to high local concentrations of growth factor, as well as the multivalency and inhibition of down regulation by the bound growth factor. A comparison of the results obtained for the bound and soluble growth factors revealed that the bound growth factors led to a much higher local concentration of growth factor in contact with the cells, which could induce multi- valency effect, such as enhancing the dime- rization of the growth-factor receptors. In addition, this effect would be further enhanced by inhibiting the internalization of the growth factor, with the bound growth factor resulting in higher levels of growth enhancement compared with the soluble material. Actually phosphorylation of the IGF receptor (IGFR) occurred rapidly and decreased in the presence of soluble IGF,whereas phosphorylation occurred gradually and was main- tained in the presence of bound IGF, even after 12 h incubation (Figure 5 b). The long activation period of bound IGF-1 is considered to be due to the inhibition of cellular internalization of bound IGF-1, thereby inducing growth enhancement over a sustained period as shown in the Supporting Information (Figure S13). The mechanism of bound growth factors, long-lasting signal transduction has been confirmed by some researchers.[11] Based on these results, we concluded that the increase in cell activity observed for the bound IGF-X could be attributed to a combination of these factors.