δ-Catenin is phosphorylated by Cdk5 in vivo on serines 300 and 357. A, Representative images to show detection of Cdk5-mediated phosphorylation of δ-catenin in Cdk5 knock-out (KO) and wild-type (WT) embryonic lysates using the commercially produced phospho-specific antibody (anti-phospho-Ser^300/357 δ-catenin). Two different amounts of δ-catenin lysates were used and immunoblotted for total δ-catenin (top). The same nitrocellulose membrane was immunoprobed for phospho-δ-catenin (middle). Identical amounts of lysates were immunoprobed using preimmune serum (bottom). B, Western blot of HEK293 cell lysates transfected with δ-catenin^Wt + p35 + Cdk5 or p35 + DNCdk5 (top) and δ-catenin^Ala + p35 + Cdk5 as well as δ-catenin^Wt + p35 (bottom) and immunoprobed for phospho-δ-catenin. It was clear that the Ala mutant was not immunodetected by phospho-δ-catenin antibody, whereas the presence of p35 that could complex with endogenous Cdk5 in these cells was sufficient to phosphorylate δ-catenin. C, Cortical neurons transfected with GFP-tagged δ-catenin^Wt (a–c) and phospho-deficient mutant (δ-catenin^Ala) (d–f) and immunodetected using the anti-phospho-Ser^300/357 δ-catenin (red). Arrow indicates transfected neurons, and the respective nontransfected neurons are indicated by arrowhead. The δ-catenin^Wt-transfected neurons showed a greater signal with the phospho-δ-catenin antibody. In contrast, the transfection of δ-catenin^Ala exhibited no increase in staining. Scale bars, 10 μm. D, Cortical neurons transfected with a DNCdk5 construct; the representative figure shows that the DNCdk5-overexpressing neuron (red) did not exhibit phospho-δ-catenin staining (green). Arrow indicates DNCdk5-transfected neuron, and arrowhead indicates the nontransfected neuron. Scale bars, 10 μm. All figures are representations of at least three independent experiments.