Gephyrin regulates KCC2 membrane expression and clustering in Neuro2a cells. A, Representative confocal maximum projection images of Neuro2a cells cotransfected with plasmids encoding either GFP or GFP-tagged gephyrin (Gphn-GFP) and a plasmid encoding Flag-tagged KCC2. Cells were immunostained for surface and total KCC2 using anti-Flag antibodies. Right, Single confocal image showing overlay of surface Flag (red) and GFP (green) immunostainings. Note the colocalization of surface Flag-KCC2 and GFP-gephyrin immunofluorescent clusters (arrowheads). Scale bar, 5 µm. B, Summary boxplots showing normalized surface/total Flag immunofluorescence intensity ratios (GFP, n = 18; GFP-gephyrin, n = 20 cells; three independent experiments). **p < 0.01, Mann–Whitney rank-sum test.