Fig. 1
From: Functional mapping of brain synapses by the enriching activity-marker SynaptoZip
Design and characterization of SynaptoZip. a Cartoon depicting the reporter system; box diagrams illustrate SZ moieties. S1, S2: spacer sequences; Zip: Acid-p1. b WB of transfected (GZ, SZ) and non-transfected (Ctrl) Hela cells, detection with anti-VAMP2, anti-Myc epitope tag, anti-GFP antibodies, and with SB-Alexa647 (see also Supplementary Fig. 1a). c Analysis of Zip-Bond binding in live Hela cells (the red line is the logistic fitting, adj. R 2 = 0.98, K d = 5.09 nM; N 0.1nM = 38, N 1nM = 65, N 2nM = 45, N 3nM = 80, N 5nM = 59, N 10nM = 39, N 30nM = 48, N 100nM = 36; mean ± s.e.m.). d SB uptake in a mixture of transfected and non-transfected Hela cells (green, eGFP; red, SB and DIC image; SB 5 nM, 1 h incubation). e Histograms of SB fluorescence and their cumulative representation (Inset; N = 320, non-expressing cells; N = 636, GZ-expressing cells; SBexpr = 0.284 ± 0.089, SBnon-expr = 0.063 ± 0.014, mean ± SD; p < 0.01, KS test). f Analysis of the fate of internalized SB in SZ-expressing Hela cells. Left, internalized SB-Alexa647 was not displaced by excess of SB-Alexa488 (time-lapse imaging, N = 6; mean ± s.e.m.). Right, vesicular SB remains up to 48 h from extracellular washout (N 5min = 71, N 0.5h = 60, N 1h = 47, N 1.5h = 60, N 2.5h = 53, N 3.5h = 73, N 5.5h = 42, N 6.5h = 39, N 21h = 37, N 24h = 50, N 48h = 47; mean ± s.e.m.). g Hela cells expressing non-fluorescent SZ were incubated sequentially with SB-Alexa488, SB-Alexa647, SB-Alexa568. h Cumulative histograms of cell fluorescence from N = 92 cells as in g, illustrating the labeling similarity among the epochs (p ≥ 0.26; KS test). Scale bars, 10 μm, 1 μm inset (d) and 10 μm (g)
