FIGURE 6.

Activities of ^I1–9, ^I6–9, and ^I1–5 for binding the ^III1–2/anastellin aggregate and inhibiting FN matrix formation. A, for the pelleting assay, 2 μm ^I1–9, ^I6–9 (∼40 kDa), or ^I1–5 (∼30 kDa) was mixed with 40 μm anastellin, with or without 10 μm ^III1–2. A significant amount of ^I1–9 co-precipitated with the ^III1–2/anastellin aggregate. On the other hand, only a trace amount of ^I1–5 co-precipitated, and ^I6–9 did not co-precipitate at all. None of the fragments formed any aggregates with anastellin in the absence of ^III1–2. S, supernatant; P, pellet; Std, BenchMark protein ladder (Invitrogen). B, various FN fragments (1 μm) were added to FN(^−/−) cell culture with FN-YPet (30 nm) and cultured for ∼16 h. ^I1–9 inhibited FN matrix formation, ^I6–9 had no significant effect on FN matrix assembly, and ^I1–5 reduced the FN matrix. C, the amount of matrix FN was estimated by measuring YPet fluorescence after solubilizing YPet from cell culture by trypsin treatment. The emission intensity was normalized to that of a control sample (no fragment). ^I1–9 and ^I1–5 reduced the FN matrix by 50 and 20%, respectively. The error bars indicate standard deviation. *, p < 0.01.