Antibody-induced integrin clustering and its effect on FAK activation. (A) Confocal images of integrin clustering induced by antibody. The stably transfected cells grown on Delta T dishes were stained with nonfunctional anti-αIIbβ3 mAb, followed by staining with goat anti–mouse IgG conjugated with Alexa Fluor 488 after (i) or before (ii) fixation. Scale bar represents 10 μm. (B) WT or α968C/β693C mutant CHO-K1 cell transfectants in suspension were incubated with (lanes 2, 3) or without (lane 1) noninhibitory anti-αIIbβ3 mAb in the absence (lane 2) or presence (lane 3) of goat anti–mouse IgG at 37°C for 1 hour. As a control, cells were incubated with ligand-mimetic antibody PAC-1 (lane 4) in the presence of 1 mM Mn²⁺ and 10 μg/mL activating antibody PT25–2 or adhered on Fg-coated dish (lane 5) in the absence (for WT) or presence (for α968C/β693C) of 1 mM DTT. FAK was immunoprecipitated from the cell lysate and blotted with antiphosphotyrosine mAb 4G10 (FAK-pY) and reblotted with anti-FAK (total FAK).