Journal: Communications Biology
Article Title: The endosomal Q-SNARE, Syntaxin 7, defines a rapidly replenishing synaptic vesicle recycling pool in hippocampal neurons
doi: 10.1038/s42003-021-02512-4
Figure Lengend Snippet: a Double immunostaining of Stx7 and Syp in cultured hippocampal neurons at 14 DIV. An upper panel shows representative axonal localization of Stx7 (green) and Syp (red). Magnified images of numbered areas (1−3) are shown individually below. The specificity of the Stx7 antibody was confirmed in independent experiments where Stx7 expression level was reduced by specific shRNA (Supplementary Fig. ). Scale bars indicate 2 µm. b Triple-immunofluorescence for Stx7 (blue), Syp (red), and an active zone maker Bassoon (BSN, green). An upper panel shows representative images. Magnified images of the numbered areas (1−3) are shown below. Scale bars indicate 1 µm. c Immunoelectron micrographs of SypHy (left) and Stx7-SEP (right) at presynaptic terminals. Immunogold labeling was intensified by silver enhancement. Arrowheads indicate both edges of the active zone deduced from postsynaptic density structures. Scale bars indicate 100 nm. d Number of immunoparticles as a function of the area of presynaptic varicosity. Sixteen varicosities for SypHy (black) and 24 varicosities for Stx7-SEP (red) were analyzed. e A box-whisker plot showing densities of SypHy immunoparticles (black) and Stx7-SEP immunoparticles (red) calculated from ( d ). p < 0.0001 with unpaired t -test with Welch’s correction. f A cumulative plot of distances of immunoparticles to the nearest AZ membrane. Note that vertical lines from the edges of AZs were drawn manually, and only immunoparticles inside the enclosed areas were measured for the analysis (Supplementary Fig. 9). Statistical significance was evaluated with Kolmogorov−Smirnov test ( p = 0.0003). g A representative quantification of Stx7 in native SVs purified from rat brains. Various amounts of recombinant GST-Stx7-N-terminal domain (GST-Stx7-N) and a fixed amount of purified SV fraction (vesicle concentration, 26.7 nM; protein concentration, 99.7 ng/μL) were subjected to quantitative western blot analysis (see also Supplementary Fig. for complete datasets and control experiments for Syb2). h Signal intensities of bands were quantified and plotted as a function of moles of GST-Stx7-N. A red circle indicates the signal intensity measured for 1.0 µg SV shown in ( g ).
Article Snippet: Cells were incubated with rabbit polyclonal anti-GFP antiserum and mouse monoclonal anti-Syb2 antibody (Cl69.1) (kind gifts from Reinhard Jahn) (Figs. b, e, and Supplementary Fig. ), or with rabbit polyclonal anti-Stx7 antibody (Bethyl Laboratories; A304-512A), mouse monoclonal anti-Synaptophysin antibody (Cl7.2), guinea pig polyclonal anti-Synaptophysin antibody (Synaptic Systems; 101 004), and mouse monoclonal anti-Bassoon antibody (Novus Biologicals; SAP7F407)(Fig. ), or chick polyclonal anti-MAP2 antibody (1:1,000; Abcam; ab5392) and anti-Stx7 antibody (Supplementary Figs. , ), for 1 h at RT.
Techniques: Double Immunostaining, Cell Culture, Expressing, shRNA, Immunofluorescence, Labeling, Whisker Assay, Membrane, Purification, Recombinant, Concentration Assay, Protein Concentration, Western Blot, Control
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