Requirement for interaction between Fer and p120ctn to regulate β-catenin phosphorylation and synaptic vesicle localization. (A and B) Hippocampal neurons from postnatal day 0 p120ctn^fl/fl mice were transfected with vector or Cre recombinase together with synaptophysin-GFP (Syn-GFP) at 10 DIV and were fixed at 14 DIV for imaging. Representative synaptophysin-GFP patterns are shown in A, and normalized coverage of synaptophysin-GFP in each condition is shown in B. n = 9 (vector) and 11 (Cre). (C–F) Imaging at 13 DIV of rat hippocampal neurons that were transfected at 10 DIV with synaptophysin-GFP (C and D) or GFP–Bsn95-3938 (E and F). Cultures were incubated with 5 μg/ml FerCo or FerP from 11 to 13 DIV. The coverage (micrometers of GFP per 10 μm of axon) of synaptophysin-GFP (n = 10 [FerCo] and 12 [FerP]) or GFP–Bsn-95-3938 (n = 8 [FerCo] and 9 [FerP]) is quantified in D and F. (G and H) Time-lapse analysis at 13 DIV of synaptophysin-GFP–expressing organelles in hippocampal neurons transfected at 10 DIV. Cultures were treated with FerCo or FerP for 2 h before time-lapse imaging. Pictures were taken every 1 min. Increased movements of puncta are observed in FerP-treated neurons compared with FerCo-treated control neurons. The average speeds of individual synaptophysin-GFP puncta in each condition are shown in G, and a histogram representing the distribution of the speeds of each puncta is shown in H. n = 71 (FerCo) and 68 (FerP). (I) 12 DIV hippocampal neurons were treated with FerCo or FerP for 4 h followed by IP with anti–β-catenin (β-cat) and immunoblotting (IB) with antiphosphotyrosine mAb 4G10 (top). The blot was reprobed with anti–β-catenin (bottom). FerCo, control peptide; FerP, Fer peptide. *, P < 0.05; **, P < 0.01; ***, P < 0.0001. Error bars represent SEM. Bars, 5 μm.