Panel A shows a series of western blot images displaying the expression levels of various synapse proteins in control, Endophilin-A1-knockdown (KD), and Endophilin-A1- and Endophilin-A2-KD rat hippocampal neurons. The proteins analyzed include Endophilin-A1, Endophilin-A2, CAST, ELKS, Munc13, Rim1, RimBP2, Synaptophysin, Dynamin-1, Homer1, and Tubulin. Panel B presents a bar graph quantifying the results from Panel A. The y-axis represents the protein expression levels normalized with Tubulin and further normalized by control neurons. The x-axis lists the proteins analyzed. The graph compares the protein expression levels in Endophilin-A1-KD and Endophilin-A1- and Endophilin-A2-KD neurons, with significant differences indicated by asterisks. Panel C shows another series of western blot images examining the effect of the Endophilin-A1-E264A mutant on active zone protein expression levels. The proteins analyzed include Endophilin-A1, Endophilin-A2, CAST, Rim1, and Tubulin in control, Endophilin-A1- and Endophilin-A2-KD, Endophilin-A1- and Endophilin-A2-KD with Endophilin-A1-WT, and Endophilin-A1- and Endophilin-A2-KD with Endophilin-A1-E264A neurons. Panel D presents bar graphs quantifying the results from Panel C. The y-axis represents the protein expression levels of CAST and Rim1 normalized with Tubulin and further normalized by control neurons. The x-axis lists the different conditions analyzed. The graph compares the protein expression levels in the different conditions, with significant differences indicated by asterisks.
Endophilin-A is necessary for stabilizing active zone proteins. (A) Protein expression levels of synapse proteins in endophilin-A-knockdown neurons. Endophilin-A1, endophilin-A2, CAST, ELKS, Munc13, Rim1, RimBP2, synaptophysin, dynamin-1, Homer1, and tubulin were detected by western blot analysis in control, EndA1-KD, or EndA1-KD and Endophilin-A2-KD rat hippocampal neurons. (B) Quantification of the results of (A). Protein expressions in EndA1-KD, or EndA1-KD and Endophilin-A2-KD neurons were normalized with tubulin and further normalized by control neurons. Results are mean ± SD. n = 3 independently prepared samples. P values were obtained by comparison with control neurons (nonparametric Mann–Whitney test, *P < 0.05). (C) Effect on active zone protein expression levels in the endophilin-A1–E264A mutant. Endophilin-A1, endophilin-A2, CAST, Rim1, and tubulin were detected by western blot analysis in control, EndA1-KD and endophilin-A2-KD, EndA1-KD and endophilin-A2-KD with endophilin-A1-WT, or EndA1-KD and endophilin-A2-KD with endophilin-A1–E264A in rat hippocampal neurons. (D) Quantification of the results of (C). CAST and Rim1 expression levels were normalized with Tubulin and further normalized by control neurons. Results are mean ± SD. n = 3 independently prepared samples. P values were obtained by comparison with EndA1-KD and endophilin-A2-KD (one-way ANOVA with post hoc Bonferroni’s multiple comparison test, **P < 0.01, ***P < 0.001). Source data are available for this figure: SourceData F10.