Fig. 4.

Integrin αV/β3 signals to Focal Adhesion Kinase via interaction with Src and PI3K. a, b T-47D breast cancer cells were exposed to T3 (1 nM) for 20 min in the presence or absence of the specific antagonist Tetrac (10 μM); Tyr³⁹⁷-FAK was analyzed through Western blot assay. Phospho-FAK densitometry values were adjusted to FAK intensity and normalized to the control. c Unlabeled T3 displace [¹²⁵I]T3 from T-47D plasma membranes. [¹²⁵I]T3 was added to purified plasma membrane proteins (60 μg/sample) before the addition of unlabeled T3 (0.1 to 1000 nM). Unlabeled T3 was effective in the displacement of radiolabeled T3 conformed best to the bind with the integrin αvβ3 in breast cancer plasma membranes. d, e Breast cancer cells were exposed to 1 nM T3 for 20 min and the cell protein extracts immunoprecipitated with an antibody vs. integrin αV/β3 (d) and FAK (e). IPs were assayed for co-immunoprecipitation of Src, p85α, FAK, and integrin αV/β3. Membranes were re-blotted for the immunoprecipitated protein to show equal loading. Protein extracts without immunoprecipitation (input) were used to identify the correct band. f Cells were incubated in the presence of T3 for 20 min in baseline conditions and co-localization of integrin αV/β3 vs. FAK was measured with immunofluorescence after staining of FAK with FITC and integrin αV/β3 with Texas Red. Nuclei were counterstained with DAPI. Experiments were performed in triplicate; representative images are shown