Figure 1: PtdIns5P activates Rac1 and Cdc42.
From: Phosphatidylinositol 5-phosphate regulates invasion through binding and activation of Tiam1
(a) MEF Tet-Off IpgD, Inp54p, or IpgD-CS-expressing cells were transfected or not with EGFP-PIP4KIIß. After serum starvation, PtdIns5P levels were quantified by mass assay. Data are shown as mean±s.e.m. (n=3–6; *P<0.05 using non-parametric t-test) (b) Lysates from serum-starved MEFs were subjected to pull-down experiments to monitor the levels of active Rac1, Cdc42 and RhoA. IpgD and Inp54p were detected using an anti-myc antibody and PIP4KIIß using an anti-GFP antibody. (c) Lysates from serum-starved MEFs were subjected to pull-down experiments to monitor levels of active Rac1 and Cdc42 as in (b). (d) Lysates from serum-starved MEFs, treated with the indicated exogenous-PtdInsP (15 μM) for the indicated times, were subjected to pull-down experiments as in (b). Data are representative of three to five experiments. (e) RPE-1 cells were serum-starved overnight and stimulated with 200 ng ml−1 FGF-1 for the indicated time and PtdIns5P levels were quantified by mass assay. Data are shown as mean±s.e.m. (n=4; *P<0.05 using non-parametric t-test). (f) Rac1 activation was monitored by pull-down from serum-starved RPE-1 cells transduced by GFP-3xPHD wild type (WT) or GFP-3xPHD PI binding mutant (Mut)-expressing lentiviruses and treated or not with 200 ng ml−1 FGF-1 for 15 min. Data are shown as mean±s.e.m. (n=3; *P<0.05 using one sample t-test for the deviation from mean value of 1 in the control group). (g) PtdIns3P levels (percentage of total PtdInsPs) in RPE-1 cells stimulated with 200 ng ml−1 FGF-1. Data are shown as mean±s.e.m. (n=3; *P<0.05 using non-parametric t-test). (h) PtdIns(3,5)P2 levels (percentage of total PtdInsP2s) in RPE-1 cells stimulated with 200 ng ml−1 FGF-1. Data are shown as mean±s.e.m. (n=3; *P<0.05 using non-parametric t-test).
