FIGURE 2.

CdGAP preferentially associates with phosphatidylinositol (3,4,5)-trisphosphate. A, MLVs composed of phosphatidylcholine or of a mix of 90 mol% phosphatidylcholine (PC) with 10 mol% of PI, phosphatidylinositol (3)-phosphate (PI(3)P), phosphatidylinositol (4)-phosphate (PI(4)P), phosphatidylinositol (5)-phosphate (PI(5)P), phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P₂), or phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃) were incubated with GST-CdGAP(1–221) in a MLV flotation assay. MLV-associated proteins were separated from unbound proteins by sucrose gradient centrifugation. Unbound and bound proteins were subjected to SDS-PAGE and analyzed by Western blotting with GST-specific antibodies (U, unbound; B, bound). B, quantitative analysis of blots shown in A, representing the bound-unbound ratios. The value obtained for PC MLVs was set to 1 and used as the reference point. Values represent an average of at least three independent experiments for each condition; error bars, S.E. *, p < 0.001 for the comparison to the reference point (PC MLVs). C, MLVs composed of PC or PC:PI(3,4,5)P3 were incubated with GST, GST-CdGAP(1–221), or GST-BMX-PH in MLV flotation assay.