(A) Loss of focal adhesion structures (white arrows) mediated by AFAP-110 downregulation. Immunofluorescence staining of vinculin, a cytoskeletal protein localized at focal adhesion structures, was performed using a monoclonal anti-vinculin primary antibody and a goat anti-mouse Alexa Fluor 594–conjugated secondary antibody (red). Actin was stained by Alexa Fluor 488–conjugated phalloidin (green). Representative images of 2 independent experiments are shown; scale bars: 20 μm. (B) Expression and activity of Src in phosphorylating adhesion-associated substrates. Western blotting of cell lysates was performed with monoclonal mouse antibodies selective for phospho-FAK tyrosine 861 (Y861), phospho–c-Src tyrosine 418 (Y418), phospho-paxillin tyrosine 118 (Y118), as well as antibodies recognizing total protein Src, FAK, paxillin, and vinculin. Cell lysate from PC3 cells that express a constitutively active form of Src (PC3 Src 527F) was used as a positive control. Membranes were probed for β-actin as loading control. Results from 1 of 2 independent experiments are shown. (C) Immunoblotting with antibodies against AFAP-110, integrin β1, and E-cadherin. β-Actin expression was used as a loading control. (D) Immunofluorescence staining of integrin β1 was performed as described in Methods, using a monoclonal anti–integrin β1 primary antibody and a goat anti-mouse Alexa Fluor 594–conjugated secondary antibody (red). Images were converted to grayscale pictures for best visualization of the localization of integrin β1 at focal adhesion structures in PC3 and scrambled control cells (yellow arrows). Representative images of 2 independent experiments are shown; scale bars: 20 μm.