Supplementary Figure 4: Separation of PIPKIα and PI3K binding on IQGAP1 attenuates PI3,4,5P3 synthesis.
(a) Schematic representation of uncoupling of PI4,5P2 and PI3,4,5P3 synthesis by inserting the 17 aa indicated between the WW and IQ domains. (b) Iqgap1 knockout (Iqgap1−/−) mouse embryonic fibroblasts (MEFs) were reconstituted with the indicated GFP-tagged human IQGAP1 constructs. Cells were treated with 10 ng ml−1 EGF for 15 min and cellular PI3,4,5P3 contents were measured by a competitive ELISA. The graph is shown as mean ± s.d. of n = 3 independent experiments. Unpaired Student t-tests were used for statistical analysis (∗, P < 0.05; ∗∗, P < 0.01; n.s., not significant). (c) Using cell lysates from the reconstituted MEFs, IQGAP1 proteins were IP’ed with an anti-GFP antibody and associated proteins were analyzed by IB. (d,e) The reconstituted MEFs were transfected with constitutively active Akt1 or PDK1 and Akt1 or PDK1 was IP’ed and the associated IQGAP1 proteins were analyzed by IB. The experiments described above were performed independently at least three times. Source data for b can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplementary Fig. 7.
