Fig. 7. N-terminal and C-terminal fragments of kinectin associating with FN-coated beads. Schematic diagram depicting full-length and various portions of kinectin tagged with VSV (A). VSV-immunoblots of the kinectin (KNT) fragments found to cluster with FN-coated beads (B) and expression levels of each kinectin fragment by NIH3T3 cells (C); indicated on the right are the molecular masses (kDa) of marker proteins. (D) Immunofluorescence staining of HFF transfected with VSV empty vector (a), VSV-kinectin fragments I, II, III and IV (b,c,d,e, respectively) and full-length kinectin (f). Integrins in cells expressing the different constructs were clustered with FN-coated beads. After 20 minutes incubation with the beads, the cells were fixed and stained with a monoclonal antibody to VSV. Cy3-conjugated goat anti-mouse IgG was used for detection of the bound antibodies. Each inset shows a higher magnification focusing on the equator of the bead marked by arrowhead. For full-length VSV-kinectin, 52% of the beads were scored as positive (see Materials and Methods for the semi-quantitative criteria); fragment I was 39% positive; fragments II and III were 9% and 8% positive, respectively; fragment IV was 53% positive; and fragment V (containing three-quarters of kinectin) was 82% positive. The mean values for induction of kinectin localization by fragments II and III were confirmed by ANOVA to be significantly lower than full-length kinectin (P<0.01), whereas the values for fragments I, IV and V were not significantly different statistically (P>0.05). Bar, 20 µm.

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