Screening for a molecule mediating the association of DNT with the FN network. (A) Profile of Mono Q anion-exchange chromatography of the culture supernatant of FN-null cells. (B) The association of DNT with the FN network of MRC-5 cells supplemented with each fraction from the chromatography. MRC-5 cells seeded in a 24-well plate were incubated overnight with eluted fractions. The next day, the cells were treated with 2 μg/ml of DNT, and stained with anti-DNT polyclonal antibody as described in Methods. Bar, 5 μm. (C) Each fraction from the chromatography was subjected to SDS-PAGE followed by silver staining. The arrows and arrowheads indicate the proteins identified by mass spectrometry. The asterisk indicates contaminated human keratin. (D) The fractions from chromatography with the culture supernatant of MC3T3-E1 cells. Nidogen-2 was detected at approximately 200 kDa, and the smaller variants of nidogen-2 are presumed to be N-terminally truncated, based on the results of mass spectrometry (arrowheads). Note that the band indicated by open arrowhead is present in fraction 4 inducing the association of DNT with the FN network.