Fig. 3

Inhibition of calcium-induced PS1-Syt1 interactions in primary neurons using PS1-LNT peptide. a The PS1-Syt1 proximity in neurons pre-treated with PS1-LNT or scramble peptide at the basal (KCl(-)) conditions or after 15-min depolarization with 50 mM KCl was determined by FLIM. The neurons were immunostained with anti-PS1 N-terminus (NT) and anti-Syt1 antibodies, followed by fluorescently conjugated secondary antibodies. The change in FRET efficiency was used to estimate relative change in the proximity between PS1 and Syt1. The pseudo-colored image presents the lifetime of the donor fluorophore in picoseconds. The orange-red pixels indicate shorter lifetimes reflecting closer proximity between the fluorescently labeled PS1 and Syt1; scale bar 10 μm. The graph shows normalized FRET efficiency between PS1 and Syt1. Note, PS1-LNT-mediated inhibition of the calcium-induced PS1-Syt1 interaction in neurons. The data are presented as mean ± SEM, n = 64 for scramble KCl (-), n = 70 for PS1-LNT KCl (-), n = 68 for scramble KCl (+) and n = 70 for PS1-LNT KCl (+), n = total number of neurons 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 shown in the Additional file 1. b In vitro IMAC analysis demonstrates successful inhibition of the PS1 binding to Syt1 by the PS1-LNT peptide. His-V5-Syt1 expressed in PS DKO cells was purified and immobilized on the beads using immobilized metal affinity chromatography, IMAC. The beads were incubated with mouse brain lysate in the presence of scramble or PS1-LNT peptide. The blue arrow indicates bands corresponding to the Syt1 immobilized on the beads. The red arrow shows the band corresponding to PS1 CTF bound to the immobilized Syt1 in the presence of scramble peptide only. Gapdh immunobloting was used as control. n = 3 independent experiments