Fig. 5. LOS induces the activation of Rac1 GTPase and PI3K. HBMECs, transiently transfected with wild-type Rac1 coupled to GFP (GFP-Rac1), with the GTPase-binding domain of PAK1 coupled to GFP (GFP-PAK1-PBD) or with the PH domain of Akt coupled to GFP (GFP-Akt-PH) were infected for 3 hours with either the 2C43 wild type (WT) or the isogenic rfaC (rfaC) or lgtA (lgtA) defective mutants of N. meningitidis. When indicated (WT + wortmannin), HBMECs were pretreated for 2 hours with 100 ng ml^–1 wortmannin and then infected with the 2C43 wild-type strain of N. meningitidis in the presence of the inhibitor. (A-C) Cells were then stained for bacteria or ezrin (red) and recruitment of the different GFP-tagged proteins at bacterial entry sites was analysed by confocal microscopy. (right) Merged images (overlay) of the same fields. (D) The frequency of recruitment of the different GFP-tagged proteins at the entry site of WT, rfaC or lgtA bacterial strains was determined by counting 50 cells. Average values (± s.e.m.) are presented from three independent experiments.