Morphologic characterization of eGFP-gephyrin and its mutant variants. A, Representative images of primary hippocampal neurons cotransfected with eGFP-WT, eGFP-S268E, eGFP-S270A, or eGFP-DN and shRNA-3′UTR. eGFP-gephyrin clusters (green), α2 GABAARs (red), and VGAT (blue) are shown. Scale bar, 10 μm. B, Quantification of eGFP-gephyrin cluster density, cluster area, and intensity shows larger eGFP-S270A clusters compared with eGFP-WT at synapses. S268E: WT n = 66 cells, S268E n = 60 cells, 4 cultures. Syn: Cluster Number (Nb) p = 0.42, area p = 0.22, intensity p = 0.05. Extra: Nb p = 0.99, Area p = 0.66, Intensity p = 0.44. S270A: WT n = 86 cells, S270A: n = 74 cells, 6 cultures. Syn: Nb p = 0.77, Area p = 0.02, Intensity p = 0.02. Extra: Nb p = 0.39, Area p = 0.42, Intensity p = 0.15. C, Quantification for α2 GABAAR clusters shows significantly more receptors in eGFP-S270A mutant clusters. S268E: WT n = 52 cells, S268E n = 47 cells, 3 cultures. Syn: Nb p = 0.48, Area p = 0.46, Intensity p = 0.6. Extra: Nb p = 0.46, area p = 0.63, intensity p = 0.22. S270A: WT n = 52 cells, S270A n = 39 cells, 3 cultures. Syn: Nb p = 0.56, Area p = 0.08, Intensity p = 0.008. Extra: Nb p = 0.008, Area p = 0.81, Intensity p = 0.29. Data shown as mean ± SEM. Values were normalized to the corresponding control values. Statistics *, p ≤ 0.05, **, p ≤ 0.01 (Mann–Whitney rank sum test).