β-Catenin overexpression suppresses Fer-mediated synaptic vesicle delocalization. (A) Phosphorylation of β-catenin (β-cat) is increased after inhibition of Fer function. 7 DIV rat cortical neurons were infected with lentiviruses expressing WT Fer or KDFer. At 14 DIV, extracts were prepared and used for IP with anti–β-catenin followed by immunoblotting (IB) with antiphosphotyrosine antibody 4G10 (left), and the blots were reprobed with anti–β-catenin (middle). Total lysates were immunoblotted with anti-Fer to examine the expression of exogenous Fer (right). (B–E) Expression of WT or Y654F β-catenin prevents synaptic puncta dispersion caused by inhibition of Fer. Hippocampal neurons were transfected with synaptophysin-GFP (Syn-GFP) together with the indicated DNAs at 10 DIV and were fixed at 14 DIV for imaging. Representative images are shown in B and C. Normalized coverage of synaptophysin-GFP per 10 μm of axon in each condition is quantified in D and E. In D, n = 8 (vector), 9 (KDFer), 8 (KDFer + WTβ-cat), and 7 (KDFer + Y654Fβ-cat). In E, n = 15 (vector), 21 (Fer shRNA), 11 (Fer shRNA + WTβ-cat), and 14 (Fer shRNA + Y654Fβ-cat). (F and G) Delocalization of presynaptic puncta by β-catenin knockdown was not rescued by overexpression of Fer. Hippocampal neurons were transfected with the indicated DNAs at 10 DIV and fixed at 14 DIV for imaging. Representative images of synaptophysin-GFP clusters in each condition are shown in F, and normalized coverage of synaptophysin-GFP per 10-μm axon in condition is presented in G. n = 13 (vector), 16 (β-cat shRNA), and 10 (β-cat shRNA + Fer). KDFer, kinase-dead mutant (K591R) of Fer. *, P < 0.05; **, P < 0.01. Error bars represent SEM. Bars, 5 μm.