Figure 5.

Coimmunoprecipitation of GM1 with α5β1 integrin but not TRPC5. A, N′ase-treated undifferentiated (Undiff.) and differentiated (Diff.) NG108-15 cells were lysed in 1% Brij 98 and precipitated with rabbit anti-TRPC5 Ab plus protein A–agarose beads. a, b, IB was performed with goat anti-TRPC5 Ab plus secondary Ab linked with HRP (a) or CtxB–HRP (b). Consistent with the RT-PCR result (Fig. 2), TRPC5 expressed in undifferentiated cells was depressed after differentiation. CtxB–HRP blot failed to detect coprecipitation of GM1 with TRPC5. B, The above lysate from undifferentiated cells was subjected to IP with anti-β1 or anti-α5 integrin Ab and IB with Ab against partner integrin plus HRP-linked secondary Ab (a) or CtxB–HRP (b). The latter blot showed GM1 in the migration front (arrow), indicating coprecipitation of GM1 with both integrins. Bands at ∼250 kDa in Ba represent α5β1 dimers. C, The precipitates were also extracted with chloroform/methanol (1:1, v/v), and the extracts were subjected to HTPLC and reaction with CtxB–HRP after N′ase treatment on the plate (Wu and Ledeen, 1988). PM, Lipids from the plasma membrane of NG108-15 cells; BBG, bovine brain gangliosides mixture; none, IP with beads alone. These results provide evidence for GM1 association with α5β1 integrin but not TRPC5.