Figure 6.

Fragmentation of cell-surface VLA-5 after exposure to FN120. (a) Lysates of U937 cells, surface labeled with ¹²⁵I, were immunoprecipitated by affinity-purified antibodies to α₅ and then fractionated by SDS-PAGE before radioautography. Lane 1, untreated cells; lane 2, FN120-treated cells; and lane 3, anti-α₅ immunoprecipitates of culture supernatants of radiolabeled cells, previously exposed to FN120, demonstrating the release of α₅ fragments. (b) Lysates of U937 cells were immunoprecipitated with rabbit antibody to β₁, fractionated, blotted to nitrocellulose, and then probed with affinity-purified rabbit antibodies to the cytoplasmic domain of α₅ (lanes 2 and 3). Lane 1, lysates incubated only with the secondary antibody; lane 2, proteins from untreated cells; lane 3, proteins from FN120-treated cells in lanes 1 and 3. An arrow indicates that the 60-kDa fragment was more prominent after FN120 treatment. (c) U937 cells cultured with or without FN120. Lanes 1 and 3, supernatants from untreated cells; lanes 2, 4, and 5, supernatants from FN120-treated cells. Immunoblots probed with second antibody only (lanes 1 and 2), with anti-β₁ antibodies (lanes 3 and 4), or with antibodies to the cytoplasmic domain of α₅ (lane 5). Note the 60-kDa fragment identified with anti-α₅ antibodies. (d) Serial densitometric measurements of a 60-kDa α₅ fragment in immunoblots of cardiac lymph collected at intervals before occlusion and for 48 hours after reperfusion in a dog with significant myocardial ischemia. (e) Immunoblot of cardiac lymph collected 5 hours after reperfusion of ischemic myocardium. Lane 1 was developed with only second antibody; lane 2 with anti-β₁; and lane 3 with antibodies to the cytoplasmic domain of α₅. The arrow beside lane 3 indicates a prominent 60-kDa fragment detected by anti-α₅ antibodies.