Figure 3.

Dynamic interaction with matrix proteins is essential for shear-induced α_vβ₃–Shc association. (A) HUVECs were allowed to adhere to FN-coated slides for 2 h in the absence of serum and then subjected to the following treatments: Lanes 1 and 2, untreated samples; lanes 3 and 4, incubated with 20 μg of the nonblocking 11E5; lanes 5 and 6, incubated with 3B8, which blocks the α₅β₁, but not the α_vβ₃, binding sites; lanes 7 and 8, incubated with 16G3, which blocks both the α₅β₁ and α_vβ₃ binding sites. HUVECs were either sheared (S) or kept as static control (C). The cell lysates were subjected to IP with LM609, and the immunoprecipitated α_vβ₃ was subjected to IB with an anti-Shc pAb. (B) Experiments were similar to Fig. 3A, except that anti-α₅β₁ mAb 1950 was used for IP. (C) Experiments were similar to Fig. 3A, except that HUVECs were seeded on VN-coated slides and incubated with either the nonblocking antibody 443 (lanes 1 and 2) or antibody 661 (lanes 3 and 4), which blocks the α_vβ₃ binding sites of VN. Gels are representative of three separate experiments.