Fig. 5. The insulin-mediated formation of PtdIns-3-P occurs through the activation of TC10. (A) L6 cells were transfected with myc-TC10 Q75L, myc-Cdc42 L61 or the empty vector. After 24 h, cells were labelled with myo-[3H]inositol and HPLC analysis of PtdIns-3-P levels was performed as described in Materials and methods. Data are mean ± SEM of two independent experiments performed in duplicate. (B) L6 cells were co-transfected with GFP–2XFYVEHrs and either myc-TC10 Q75L, myc-Cdc42 L61 or HA-TC10 T31N. After 24 h, cells were serum starved for 6 h and analysed by confocal microscopy. HA-TC10 T31N-co-transfected cells were stimulated with 300 nM insulin for 3 min before confocal analysis. Arrows indicate the plasma membrane localization. Arrowheads mark the filopodia induced by overexpression of myc-Cdc42 L61. Bar, 10 µm. (C) L6 cells were transfected with myc-TC10 wt, myc-TC10 Q75L or myc-Cdc42 L61. Serum-deprived cells were left untreated or stimulated with insulin for 3 min. Lysates were incubated with GST-Pak1 PBD and pull-down assay was performed as described in Materials and methods. Association of the overexpressed proteins with the GST-Pak1 PBD was assessed in western blot analyses by using an anti-myc antibody (upper panels). Equal amount of the overexpressed proteins in lysates was confirmed by loading an aliquot of each lysate (bottom panel). Blot is representative of three independent experiments.