FIGURE 1.
GIV does not fractionate with Gαi3 in cell membranes. A–C, HEK293T (A), HeLa (B), or MDA-MB-231 (C) cells were homogenized in the absence of detergents, and the PNS was separated into supernatant (S100) and pellet (P100) after 100,000 × g centrifugation. P100 fractions were resuspended in buffer containing Triton X-100 and centrifuged to obtain the detergent soluble (TX-S) and insoluble (TX-I) fractions. Immunoblots (IB) of equal aliquots of each fraction from one experiment representative of three is shown. TfR and tubulin were used as membrane and cytosol markers, respectively. D, detergent lysates of MDA-MB-231 cells were centrifuged in a discontinuous sucrose gradient (5-35-45%) to separate low density detergent-resistant membranes (“lipid rafts”) from other cell fractions. Equal gradient fractions were immunoblotted with the indicated antibodies. Caveolin-1 was used as lipid raft marker, whereas β1-integrin and tubulin were membrane and cytosolic markers, respectively, of non-lipid raft fractions. One experiment representative of three is shown.