Fig. 2

Activity-dependent presynaptic recruitment of NHE6 is completely inhibited by SCAMP5 KD. a Representative images of cultured hippocampal neurons co-transfected with BFP-scrRNA or -shRNA SCAMP5, GFP-synaptophysin (SYP) and endogenous NHE6 proteins were immunostained (NHE6) after treatment of forskolin (+ cLTP) or DMSO (− cLTP). Scale bar: 5 μm. b Fluorescent intensity profiles of the respective protein along the axon. a.u., arbitrary unit. c The number of presynaptic boutons per 10 μm axon. CTL with − cLTP, 3.323 ± 0.251 counts/10 μm, n = 10 (coverslips); CTL with + cLTP, 4.282 ± 0.166 counts/10 μm, n = 7 (coverslips); SC5 KD with − cLTP, 3.451 ± 0.274 counts/10 μm, n = 10 (coverslips); SC5 KD with + cLTP, 4.572 ± 0.429 counts/10 μm, n = 7 (coverslips), F_{(3, 30)} = 4.354, p = 0.012, analyzed by one-way ANOVA test followed by LSD post hoc test. d Manders’ colocalization coefficients of SYP overlapping NHE6. CTL with − cLTP, 0.522 ± 0.039, n = 5 (coverslips); CTL with + cLTP, 0.761 ± 0.037, n = 5 (coverslips); SC5 KD with − cLTP, 0.284 ± 0.017, n = 7 (coverslips); SC5 KD with + cLTP, 0.132 ± 0.019, n = 6 (coverslips), F_{(3, 19)} = 98.777, p = 9.028E−12, analyzed by one-way ANOVA test followed by LSD post hoc test. e Percent change of presynaptic NHE6 localization extent after cLTP induction. CTL, + 45.841 ± 7.048%, n = 5 (coverslips); SC5 KD, − 53.451 ± 6.551%, n = 6 (coverslips), p = 2.8E−06, analyzed by Student’s t-test. Values are indicated as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001