The PDZ domain and protein 4.1–binding site of CASK are involved in dendritic spine formation. (A) Overexpression of CASK PDZ domain (PDZ), the C-terminal tail of syndecan-2 (synd-2C), and the CASKΔp4.1 mutant prevented spinogenesis. Cultured hippocampal neurons were cotransfected with GFP and various constructs as indicated at 13 DIV. 5 d later, cells were harvested for immunostaining using HA tag and GFP antibodies. Only the GFP patterns are shown. (B) Quantitative analysis of A. The total number of spines analyzed in each group was 756 GW1 control, 385 PDZ, 535 Synd-2C, and 494 CASKΔp4.1. More than 10 neurons for each group were analyzed. Mean ± SEM for each group is shown. (C) Overexpression of the CASK PDZ domain and CASKΔp4.1 mutant does not rescue the phenotype of CASK knockdown. At 12 DIV, cultured hippocampal neurons were cotransfected with GFP-actin and various constructs as indicated. Immunostaining using CASK, HA tag, and GFP antibodies was performed at 18 DIV. Only GFP patterns are shown. (D) Quantitative analysis of C. The total number of neurons and spines analyzed in each group was as follows: nonsilencing + GW1 vector, 31 and 2,703; nonsilencing + CASK, 27 and 4,037; shRNA + GW1 vector, 31 and 1,754; shRNA + PDZ, 32 and 1,317; and shRNA + Δp4.1, 31 and 2,085. Mean ± SEM for each group is shown. Bars, 2 μm.