Localization of the endophilin-A1 E264A variant and the roles of endophilin-A1 and endophilin-A2. (A and B) Cultured rat hippocampal neurons (control or EndA1-KD) were stained for endophilin-A1. The specificity of the endophilin-A1 antibody was confirmed by a marked reduction in fluorescence signal following EndA1-KD. Scale bar: 2 µm (B) Immunofluorescence images acquired by STED microscopy showing CAST (magenta) and endophilin-A1 (green; WT in the top, E264A mutant in the bottom) in EndA1-KD rat hippocampal neurons expressing either WT or mutant endophilin-A1. Scale bars: 1 µm. The right panels show mean fluorescence intensity profiles from 10 active zones. (C) The left panel shows immunofluorescence images of Bassoon (green) in control and EndA1/A2-KD rat hippocampal neurons. Scale bar: 2 µm. The dot plots on the right show the area of Bassoon puncta in control and EndA1/A2-KD neurons (∼10–50 puncta were analyzed per image from 20 images per group, and the mean was calculated; the experiment was repeated four times using independent cultures). Error bars indicate SD (n = 4). P values were obtained by nonparametric Mann–Whitney test. (D) Rat hippocampal cultured neurons (control or EndA1/A2-KD) were stained for synaptotagmin-1 and synaptophysin. The dot plots on the right show the correlation coefficients between synaptotagmin-1 and synaptophysin (n = 10 images per group). Scale bar: 2 µm. P values were obtained by nonparametric Mann–Whitney test.