Fig. 2

SGIP1α is expressed at the presynaptic terminals of cultured hippocampal neurons and SGIP1α KD causes the selective defects in the internalization of Syt1. a Rat brain hippocampal neurons were fixed at DIV 21 and immunostained with specific antibodies against SGIP1α (red) and Syt1 (green). Scale bar, 5 μm. b Pearson’s coefficient of colocalization between SGIP1α and Syt1 (n = 10). (c and d) Primary cultured neurons at DIV 6 were infected with AAV-shRNA-SGIP1α, and the KD efficiency was confirmed by western blotting with anti-SGIP1 antibody at DIV 21 (n = 3 blots). e-j Average Syt1-pHluorin (e, control: n = 8, KD: n = 12), synaptophysin-pHluorin (g, control: n = 14, KD: n = 17), or VAMP2-pHluorin (I, control: n = 11, KD: n = 19) fluorescence intensity profiles in neurons expressing empty vector or shRNA-SGIP1α, plotted as ΔF/F₀ against time, after stimulation with 300 APs at 10 Hz (dark bar). Fluorescence values were normalized to the maximal fluorescence signal in each experimental condition. f, h, j τ values for the decay of Syt1-pHluorin (f), synaptophysin-pHluorin (h), or VAMP2-pHluorin (j) after stimulation fitted by a single exponential. Data are means ± SEM; *p < 0.05, ***p < 0.001 by Student’s t-test, N.S., not significant