Expression of RIAM-(1–176)-CAAX targets talin to
integrins in the plasma membrane. A, A5 cells transiently
expressing mCherry-talin and GFP-tagged RIAM-(1–176)-CAAX,
RIAM-(1–176)-A-SAAX, or RIAM-(1–176) (top three panels),
or with RIAM-(1–176)-CAAX and HA-Rap1GAP (bottom
panel) were adhered to fibrinogen-coated coverslips and stained to
visualize integrin αIIbβ3 and Rap1GAP. Depicted are the
localization of αIIbβ3(blue), talin (red), RIAM
(green), or Rap1GAP (white). Images shown are maximal
projections of deconvoluted 0.1-μm z-section images of the entire cell
volume. B, A5 cells expressing HA-talin and GFP-tagged
RIAM-(1–176)-CAAX or RIAM-(1–176) were
Dounce-homogenized, fractionated, and analyzed by Western blotting for the
distribution of HA-talin and GFP-RIAM in membrane (M), cytosol
(C), and total (T) fractions with the corresponding anti-HA
or anti-GFP antibodies. In addition, integrin αIIb and RhoGDI were
immunoblotted to validate separation of the membrane and cytosol fractions,
respectively.