FIGURE 5.

PDEγ(46–87)-Btn pulls down αt-GDP-AlF₄⁻ more efficiently than αt-GTPγS. A, pulldown experiments were performed essentially as described under “Experimental Procedures.” To each 0.4 μl of streptavidin beads 0.5 μg of PDEγ(46–87)-Btn was immobilized. PDEγ(46–87)-Btn was bound completely to the beads (data not shown) and then incubated with various concentrations of αt-GDP-AlF₄⁻ or αt-GTPγS for 1.5 h at 4° C in Buffer B. After washing the beads twice with 100 μl of Buffer B, αt remaining on the beads was eluted with the SDS/DTT-containing sample buffer and detected by Western blotting using the polyclonal rabbit antibody against the N terminus of αt. The molar ratios of αt versus PDEγ(46–87)-Btn are shown under the blot. B, quantification of αt pulldown in the Western blot in A. Background in the blot was subtracted, and the intensity of each band is expressed as a percentage of the highest signal (the last lane of the upper panel). C, statistical comparison of pulldown of the two αt conformers by PDEγ(46–87)-Btn (at the molar ratio of 2 αt versus 1 PDEγ). Each bar represents a mean (±S.D. (error bars), n = 3) of percent αt pull down relative to the input. **, t test, p < 0.01.