Fig. 1. P-Rex1 localises to the shaft and distal tip of PC12 neurite growth cones. (A) Domain structure of P-Rex1 showing the Dbl homology (DH), pleckstrin homology (PH), Dishevelled, EGL-10, pleckstrin homology (DEP), post-synaptic density, disc-large, ZO-1 homology (PDZ), PEST sequences (P) and 4-phosphatase homology (4ptase) domains. The peptide sequence used as an immunogen to generate the P-Rex1-specific antibody is indicated. (B) Cell lysates from mouse brain, rat E18 hippocampal neurons and undifferentiated PC12 cells (30 µg) were immunoblotted with affinity-purified P-Rex1 antibodies. Immunoreactive endogenous P-Rex1 is indicated by the arrow. (C) PC12 cells were left undifferentiated, or NGF-stimulated as indicated, then immunostained with P-Rex1-specific antibodies (green) and Texas Red-conjugated phalloidin (red). Merged images are shown in the third column and enlarged images of the boxed areas of growth cones of immature (1) or mature (2) neurites are shown in the bottom row. The growth cone peripheral (P) and central (C) zones are indicated in the bottom row. P-Rex1 localisation at the distal tip of filopodia and lamellipodia is indicated by arrows and at growth cones by arrowheads. Undifferentiated PC12 cells were also stained with P-Rex1 peptide-adsorbed antibody as control (right-most panel). Scale bars: 10 µm.