Supplementary Figure 3: PI3,4,5P3 synthesis requires concerted PI4,5P2 generation by PIPKIα nad.
(a) PI3,4,5P3 generated by PI3K and IQGAP1 fragments from 25 μM liposomes containing 10 molar % of PI4,5P2 was measured. The graph is shown as mean ± s.d. of n = 3 independent experiments. (b) Schematic representation of canonical versus IQGAP1-mediated PI3,4,5P3 synthesis pathways. (c) The indicated PH domains were stably expressed in Hs578T cells. Cells grown in tissue culture were photographed in bright field and fluorescent channels at 200X magnification. Roughly 70–80% of cells express exogenous proteins. Scale bar, 100 μm. (d) Hs578T cells stably expressing the indicated PH domains were treated with 10 ng/ml EGF for 10 min. Cell lysates were analyzed by IB (top) and pS473Akt immunoblots of n = 4 independent experiments were quantified (middle). PI3,4,5P3 levels were measured by a competitive ELISA and the graph is shown as mean ± s.d. of three independent experiments (bottom). Paired Student t-tests were used for statistical analysis (∗, P < 0.05; ∗∗, P < 0.01; n.s., not significant). (e) Hs578T cells were stably expressed with shRNA against IQGAP1. Cells expressing non-targeting shRNA were used as a control. Cells were grown to confluence, wounded and fixed 3 h later, followed by immunostaining for PIPKIα and PI3,4,5P3. Cells were photographed at 400X magnification. Scale bar, 100 μm. (f) Immunostaining images of e were analyzed and percent of cells (over 100 cells counted for each condition) that are positive for both PIPKIα and PI3,4,5P3 signals at the leading edges were shown in the graph (n = 120 for shCon and 110 for shIQGAP1, mean ± s.d. of three independent experiments). Unpaired Student t-tests were used for statistical analysis (∗, P < 0.05; ∗∗, P < 0.01; n.s., not significant). The experiments described above were performed independently at least n = 3 times. Source data for d,f can be found in Supplementary Table 1. Unprocessed original scans of blots are shown in Supplementary Fig. 7.
