3). These result indicate that the association of DNT with FN is not related to the intoxication. When human FN was supplied to the culture, FN-null cells showed the colocalization of the toxin and FN. In contrast, DNT did not colocalize with the FN network developed on MRC-5 cells (Fig. 3). These results suggest that DNT does not interact directly with FN, and another cellular component, which is present in the culture of FN-null cells but not MRC-5 cells, is necessary for the

interaction. In fact, MRC-5 cells supplemented with the culture supernatant of FN-null cells showed the colocalization of DNT and the FN network (Fig. 4). Treatment with heat at 95°C or proteinase K abolished the ability of the culture supernatant to recruit DNT to the FN SCH772984 cell line network, which indicates that the unknown

component exists in the culture supernatant of FN-null cells and contains a protein moiety (data not shown). Figure 3 Colocalization of DNT with the FN network on various cells. Cells were incubated with DNT and stained with anti-DNT monoclonal antibody and anti-FN polyclonal antibody. FN-null cells were incubated with or without human FN (hFN) before DNT treatment. Bars, 5 μm. Figure 4 Colocalization of DNT with the FN network on MRC-5 cells supplemented with the culture supernatant of FN-null cells. MRC-5 cells, which were pre-cultured with or without the culture supernatant of FN-null cells (FN-null CS), were incubated with DNT and stained with anti-DNT monoclonal antibody and anti-FN polyclonal antibody. Bars, 5 μm. Screening for a molecule mediating Tyrosine Kinase Inhibitor Library mw the colocalization of DNT and the FN network We tried to isolate the unknown component from the culture supernatant

of FN-null cells by ion-exchange chromatography (Fig. 5A). Each fraction eluted by Mono Q anion-exchange chromatography was added to the culture of MRC-5 cells, and checked for the ability to recruit DNT to the FN network. Glycogen branching enzyme Simultaneously, each fraction was subjected to SDS-PAGE and proteins in the fractions were identified by mass spectrometry. Fraction 4 apparently induced the association of DNT with the FN network on MRC-5 cells (Fig. 5B). Mass spectrometry revealed that fraction 4 contains ECM-related proteins such as nidogen-2 in an N-terminally truncated form (open arrowhead), and lysyl oxidase-homolog 2 (LOXL2) and 3 (LOXL3) (Fig. 5C). Similar results were obtained from the culture supernatant of MC3T3-E1 cells: the truncated form of nidogen-2 (open arrowhead) and LOXL3 were found in fraction 4, which induced the association of DNT with the FN network on MRC-5 cells (Fig. 5D). LOXL2 was expressed at neither the mRNA nor protein level in MC3T3-E1 cells, which show intensive colocalization of DNT and the FN network (Fig. 3). LOXL3 supplemented to the culture did not induce the colocalization of DNT with the FN network on MRC-5 cell (data not shown).