Fig. 1
From: Dynamic presenilin 1 and synaptotagmin 1 interaction modulates exocytosis and amyloid β production
Determination of Syt1-interacting region within PS1 sequence. a Schematic presents the PS1-Syt1 complex and the 15-amino acid deletion within the PS1 L6-7 sequence (del265-279). b Deletion within the L6-7 domain of PS1 inhibits PS1-Syt1 interaction. PC12 cells transfected with FLAG-tagged wild type (wt) or del265-279 PS1 were immunostained with anti-FLAG and anti-Syt1 antibodies, followed by fluorescently conjugated secondary antibodies, and analyzed by FLIM. The graph shows normalized FRET efficiency between fluorescently labeled endogenous Syt1 and overexpressed PS1 in PC12 cells stimulated for 15 min with 50 mM KCl. The data are presented as mean ± SEM, n = 38 for PS1 wt, and n = 33 for PS1 del265-279, n = total number of cells analyzed in 3 independent experiments. Statistical significance was determined using two-tailed unpaired Student’s t-test, ***p < 0.001. The fluorescence lifetimes and corresponding FRET efficiency values are specified in the Additional file 1. c Syt1 co-purifies more efficiently with His-PS1 wt than with His-PS1 del265-279. Overexpressed His-tagged PS1 and endogenous Syt1 complexes extracted from PC12 cells following 15-min 50 mM KCl application were purified using immobilized metal affinity chromatography (IMAC) and analyzed by western blotting; unspecific bands are marked with blue asterisks. PS double knock-out (PS DKO) mouse embryonic fibroblasts (MEF) cells were used as a negative control. The adjacent graph presents quantification of the Syt1 levels co-purified with PS1. The data are presented as mean ± SEM, n = 3; two-tailed unpaired Student’s t-test, ***p < 0.001. d PS1 with the deleted exon 9 (PS1Δe9) interacts with Syt1. PS DKO and PS DKO cells stably expressing PS1 wt or PS1Δe9 were transiently transfected with His-V5-Syt1 and incubated for 15 min with 5 μM A23187 calcium ionophore. Anti-His antibody or normal mouse IgG as a control were used for the immunoprecipitation, and anti-V5 and anti-PS1 CT antibodies were applied for the immunodetection. The experiment was performed three times