α5β1 is required for recycling of EGFR1; and uncoupling EGFR1 recycling from α5β1 inhibits migration in 3D. (A–C) Cells were transfected with short hairpin vectors targeting β3, α5, β1, RCP, or EGFR1 or with truncated GFP-RCP^379-649 as indicated. The receptor recycling protocol was then followed as for Fig. 1 A but with biotinylated EGFR1 or α5β1 integrin being determined by capture ELISA using microtitre wells coated with anti-EGFR1 or α5 integrin monoclonal antibodies as indicated by the y-axis labeling of the graphs. The proportion of EGFR1 or integrin recycled to the plasma membrane is expressed as a percentage of the pool of integrin labeled during the internalization period. Values are mean ± SEM; n = 3 or 4 independent experiments. (D) Migration of A2780 cells expressing GFP, GFP-RCP^wt, or GFP-RCP^379-649 into matrigel plugs containing 25 μg/ml FN. 1 μM cilengitide (CIL) was added as indicated to the matrigel and to both the upper and lower chambers of the inverted invasion assay. Migrating cells were detected and quantified as for Fig. 4. Values are mean ± SEM for three independent experiments.