Fig. 2. Localization of PITP during neuronal maturation in hippocampal neurons. E18 hippocampal neurons were cultured for 2, 5 and 14 days before fixation and antibody labelling. (A) At day 2 (stage 3 neurons), PITP mainly localized to axonal processes (arrowhead). No or little staining was present in the remaining shorter processes clearly visible in the Phalloidin staining visualizing F-actin (red). (B) Line scan of a stage 3 hippocampal neuron demonstrating the relative intensity of PITP fluorescence along each minor neurite (n1-n4) and the axon. (C) Mean relative PITP fluorescence intensity in neurites and axonal processes. Each data point is the mean ± s.e.m., n=7. (D) Percentage of hippocampal neurons that showed PITP fluorescence in all processes (ubiquitous) or in the axon, only. Each data point is the mean ± s.e.m., n=106, ^*P<0.0001. (E) PITPβ was enriched in the Golgi region of stage 3 hippocampal neurons. (F) At day 5 (stage 4 neurons), anti-PITP antibody labelled both axonal and dendritic processes. (G) At day 14, anti-PITP immunostaining appeared punctate and showed some co-localization with the pre-synaptic marker synaptophysin (red). (H) Hippocampal neurons were cultured for indicated days in vitro (DIV), lysed and 10 µg protein was separated and analysed by western blotting using anti-PITP. Scale bars: 20 µm.