Fig. 5. PITP-induced increases in axonal length are mediated by PI3K signalling. Hippocampal neurons were nucleofected with GFP, PITP-GFP or PITP siRNA, and cultured for 48 hours on poly-lysine/laminin or poly-lysine substrates. (A) Neurons expressing PITP-GFP did not exhibit increases in axonal length when cultured on poly-lysine alone. In addition, PI3K inhibition by LY294002 (at 10 µM) antagonized PITP-GFP-mediated increases in axonal length on poly-lysine/laminin substrate. Each data point is the mean ± s.e.m. of at least three independent experiments, n=120. (B) Application of BDNF (50 ng/ml) to hippocampal neurons induces an increase in axonal length, which was partially blocked by LY294002 and fully inhibited by knockdown of PITP by siRNA. Data points show the mean ± s.e.m. of three independent experiments (n>60). (C) Cell lysates of hippocampal neurons expressing GFP, PITP-GFP or PITP siRNA were analysed by western blotting using indicated antibodies. (D) Normalized relative band density of pAkt/Akt and pAkt/actin reveal increased activity of PI3K signalling following overexpression of PITP-GFP (pAkt/actin), n=5. (E) GFP and (F) PITP-GFP expressing hippocampal neurons were fixed after 48 hours and stained using anti-pAkt antibody (red). (G) The relative fluorescence intensity of pAkt labelling in axonal growth cones of PITP-GFP expressing neurons is significantly increased when compared with GFP expressing neurons. Each data point is the mean ± s.e.m. of seven independent experiments (n>120). ^**P<0.005, ^***P<0.0001.