Figure 4.

IQGAP1 interacts with the phosphoinositides through a polybasic motif. (A) GFP-PLCδ1-PH was transiently expressed in MDA-MB-231 cells and endogenous IQGAP1 was immunostained. Cells were photographed at × 600 magnification. (B) PIP₂ liposomes (2.5%) were incubated with 0.5 μM GST-IQGAP1-N or -C for 10 min. Liposome-bound IQGAP1 was pelleted by centrifugation. Equal volume of the supernatant and the pellet were resolved by SDS–PAGE and IQGAP1 in each fraction was analysed by immunoblotting with an anti-GST antibody. (C) GST–tagged WT or deletion mutants were used for a sedimentation assay with 2.5% PIP₂ liposomes. (D) Amino acid sequence alignment of the PBM2 region among IQGAPs from the indicated species. (E) Selected lysine residues were mutated to alanines to generate a series of AA mutants (top). Binding of WT and the AA mutants to 5% PIP₂ liposomes were tested (bottom). (F) Binding of GST–tagged WT and the AA3 mutant to 5 μM of 5% phosphoinositide liposomes were tested. Samples were analysed as above and liposome-bound proteins were detected by immunoblotting with anti-GST antibody. Immunoblots were quantified and the graph is shown as mean±s.d. of three independent experiments. All the experiments described above were performed independently at least four times.

Source data for this figure is available on the online supplementary information page.

Source Data for Figure 4 ^{(249.8KB, pdf)}