Figure 3: PtdIns5P elevation activates Tiam1, which is required for actin dynamics.
From: Phosphatidylinositol 5-phosphate regulates invasion through binding and activation of Tiam1
(a) GEF trapping assays using GST-Rac1G15A agarose beads were performed on serum-starved RPE-1 cells stimulated or not with 200 ng ml−1 FGF-1 for 15 min and treated or not with NSC23766. Tiam1 and p115 RhoGEF binding were detected by western blotting. (b) Same experiment as in (a) performed on serum-starved MEF cells. (c) Same experiments as in (a) carried out on serum-starved Karpas 299 and Cost cells treated overnight or not with 15 μM WHI-154. Lysates were probed with anti-Tiam1, anti-P-NPM-ALK(Y664) or anti-ALK (Sp8) antibodies. (d) Lysates from primary WT and Tiam1−/− MEF cells were subjected to western blot analysis with anti-Tiam1. (e) Serum-starved primary WT and Tiam1−/− MEFs were treated or not with exogenous PtdIns5P (15 μM) for 30 min. Cells were then fixed and the actin cytoskeleton was visualized by staining with AlexaFluor 488-phalloidin. Scale bar, 10 μm. (f) Serum-starved primary WT and Tiam1−/− MEFs transfected with HA-Tiam1 were treated with exogenous PtdIns5P (15 μM) for 30 min. Cells were then fixed and the actin cytoskeleton was visualized by staining with AlexaFluor 488-phalloidin and Tiam1 with and HA antibody. Scale bar, 10 μm. (g) Quantitative analysis of the experiment described in (e) and (f). 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 100 in the control group and using non-parametric one way ANOVA (Kruskal–Wallis) with Dunn’s post test).
