Figure 8.

Assay for interaction between G protein subunits and the amino-terminal domain of caveolin. (A) Purified GST (upper panel) or GST-NT-caveolin containing caveolin:CH₆ residues 1–101 (lower panel) immobilized on glutathione agarose beads (3500 pmol) was incubated with purified myristoylated α_o (410 pmol), bovine brain βγ (580 pmol), or α_oβγ heterotrimer (410 pmol) as indicated at the top of the figure. After allowing the proteins to interact overnight, the resin was washed six times, and bound GST and any associated proteins were eluted with 25 mM reduced glutathione. Equal portions of the load samples (L), protein that did not bind the affinity resin and was present in the flow through (FT), the first wash (W1), the last wash (W6), and glutathione eluates (E) were analyzed for the presence of G protein subunits by immunoblotting with specific G protein α and/or β antisera. Film was exposed to the blot for 2 min. (B) Samples from glutathione eluates from each binding condition described above were analyzed side-by-side for comparison and analyzed for the presence of G protein subunits by immunoblotting. Film was exposed to immunoblots for either 2 min or, to maximize detection of the chemiluminescence signals, for 15 h. There is a hint of signal for α_o perceptible in the GST NT-caveolin eluate only after prolonged exposure of the blot to film. This signal represents an extremely small portion of that which was loaded on the resin. An ink dot was placed to the right of each of three bands detected after the 15-h exposure: two bands corresponding to β (one each for GST and GST-NT-caveolin:CH₆ elutions) and one for α_o in the elution from GST NT-caveolin:CH₆.