Fig. 4. Kinectin is markedly enriched in adhesion complexes induced by FN and FN III_5-10 fragment-coated beads. Beads coated with fibronectin (FN), a fibronectin fragment containing type III domains 5-10 (III_5-10RGD), or the same fragment with an inactivated RGD site (III_5-10KGE) were used to obtain bead-associated protein complexes (Beads) or unbound fractions (Sup). The samples were separated on a 4-12% SDS-containing polyacrylamide gel, transferred to nitrocellulose, and probed with the indicated antibodies. Note that in the beads fraction, kinectin is remarkably enriched, while kinesin and tubulin are virtually absent. In this particular experiment, 91% of total cellular kinectin was found in the beads fraction, while the corresponding percentages of total -actinin and actin in the same fraction were 22% and 23%, respectively. Also note that no proteins are observed in the unbound fractions using the functionally inactivated fibronectin fragment (III_5-10KGE), because the absence of integrin binding and adhesion to this control polypeptide resulted in no cells being isolated at the first step of the procedure where beads and bound cells are isolated using a magnetic concentrator.