Fig 1: Inhibition of exosome generation improves MI disrupted SV distribution. a Intraperitoneal injection of GW4869 prevented the synaptic vesicles accumulation at the presynaptic active zone in hippocampal neurons of MI mice. b GW4869 prevented an increase of the total SV number in the synapses in the hippocampi of MI mice. PLevene < 0.0001, one-way ANOVA: F = 19.799, P < 0.0001; Fisher’s PLSD test: Psham: MI < 0.0001, P MI:GW4869 < 0.0001. n = 20 synapses from 3 mice for each group. c GW4869 redistributed SVs at the active zones of synapses in the nerve terminals of Tg mice. 0 nm shell: PLevene = 0.669, one-way ANOVA: F = 3.424, P = 0.039; Fisher’s PLSD test: P sham: MI = 0.360, P MI:GW4869 = 0.012; 50 nm shell: PLevene = 0.016, one-way ANOVA: F = 6.703, P = 0.002; Fisher’s PLSD test: P sham: MI = 0.003, P MI:GW4869 = 0.002; 100 nm shell: PLevene < 0.0001, One way ANOVA: F = 14.785, P < 0.0001; Fisher’s PLSD test: P sham: MI = 0.0001, P MI:GW4869 < 0.0001. n = 20 synapses from 3 mice for each group. d GW4869 did not affect the length of active zone in hippocampi of MI mice. PLevene = 0.271, one-way ANOVA: F = 1.084, P = 0.345; Fisher’s PLSD test: Psham: MI = 0.185, PMI:GW4869 = 0.885. n = 20 active zones from 3 mice for each group. e Expression of SV related proteins in the hippocampi of MI mice. Left: sample bands of immunoblotting images. Right: statistical analysis of Munc 18, VAMP-2, syntaxin- 1A and SNAP-25. *P < 0.05 (f) Intraperitoneal injection of GW4869 prevented the elevation of miR-1 level in the hippocampi of MI mice, as measured by qRT-PCR. PLevene < 0.0001, one-way ANOVA: F = 7.756, P = 0.011; Fisher’s PLSD test: P sham: MI = 0.004, P MI:GW4869 = 0.018. n = 4 animals for each group. g Intraperitoneal injection of GW4869 prevented the decreased SNAP-25 expression in the hippocampi of MI mice, as measured by western blotting. PLevene = 0.265, one-way ANOVA: F = 7.647, P = 0.011; Fisher’s PLSD test: P sham: MI = 0.010, P MI:GW4869 = 0.006. n = 4 animals for each group. *P < 0.05 versus WT; #P < 0.05 versus Tg
Fig 2: SNAP25 inhibits migration and invasion of tumor cells, and inhibits dendron formation of glioma tissues. (A) Ectopic expression of SNAP25 inhibits cell migration as determined by wound healing assays. (B) Ectopic expression of SNAP25 inhibits cell migration as determined by transwell assays and analysis of representative migrated rates of glioma cells is shown below. *p < 0.05 compared with Lv-NC or sh-NC group. (C) Representative pictures of glioma cell invasion (red curved line: invaded area, blue circle: spheroid) and analysis of quantitative invaded area of glioma cells using Image J software is shown below. *p < 0.05 compared with Lv-NC or sh-NC group. (D) Glioma cell (Mcherry staining) and neuronal dendron (MAP2 staining) in the brains of indicated mice at the time of harvesting (Day 21).
Fig 3: Tomosyn is a SNAP25 binding protein in the posterior pituitary.(a) In vitro binding assay. Rat posterior pituitary lysates was incubated with GST-SNAP25 and glutathione (GSH) Sepharose. Eluted proteins bound to the beads were separated on SDS-PAGE followed by silver staining of proteins. The arrow indicates the band of interest. (b) Western blotting of pull-down samples. The arrowhead indicates the location of the immune reactive band reacting with anti-tomosyn antibody (Santa Cruz Biotechnology #sc-136105). Lane 1 = GST-SNAP25 with rat posterior pituitary lysates; lane 2 = GST-SNAP25 without rat posterior pituitary lysates (for both a, and b); Marker = molecular weight markers.
Fig 4: Tomosyn forms SNARE complexes with SNAP25 and syntaxin 1 in the posterior pituitary.(a–c) Coimmunoprecipitation assays were performed using protein G Sepharose and mouse immunoprecipitation (IP) antibodies (a), protein A Sepharose and rabbit IP antibody (b) or ImmunocruzE™ imaging system (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse IP antibody (c), respectively. Eluted samples were subjected to SDS-PAGE followed by western blotting using anti-tomosyn, anti-SNAP25, anti-syntaxin 1A, or anti-syntaxin 1B antibodies (arrowheads at right). Input was 1/200 (volume) of the total rat posterior pituitary lysates used for the assay. CTR = IP with anti-IgG antibody.
Fig 5: In the absence of endogenous αSyn, recombinant αSyn fails to multimerize(A) Semiquantitative western blotting of a known reference of recombinant WT and 3K hu αSyn (6 μM) and the electroporated synaptosomal sample. The densitometry comparison shows that the recombinant αSyn inside synaptosomes reached a concentration of ~5.5 and 6.5 μM for WT and 3K αSyn, respectively.(B) The ratio of endogenous SNAP25 (red signal in WB) to recombinant αSyn (green signal in WB) is the same in both WT and 3K experiments.(C) 1D 1H-15N-filtered spectrum of WT and 3K αSyn in αSyn-KO synaptosomes (i.e., without endogenous αSyn).(D) 2D 1H-15N HSQC NMR spectra of WT and 3K αSyn in NMR buffer (gray spectra) and inside synaptosomes purified from αSyn-KO mouse brains (blue and orange spectra, respectively). WT and 3K αSyn are N-terminally acetylated.(E) Quantification of NMR signal attenuation between “in-synaptosome” (I) and “in-buffer” (I0) signals for WT and 3K αSyn. The dashed gray line represents the expected signal for a sample made entirely of monomeric, unfolded αSyn.(F) Residue-resolved combined chemical shift perturbations (CSPs) of backbone amide resonances between αSyn “in-buffer” and “in-synaptosome” for WT and 3K αSyn (blue and orange dots, respectively). The dashed gray line marks a significance level of one standard deviation (SD) from the mean.(G) Inside synaptosomes, electroporated recombinant WT αSyn forms multimers that resemble those found in intact cells, while the 3K mutation significantly reduces their formation.(H) In the absence of endogenous αSyn, both WT and 3K recombinant αSyn fail to multimerize inside synaptosomes.(I) Quantification of the multimer-to-monomer ratio of WT and 3K αSyn inside synaptosomes with and without endogenous αSyn.In (B), a Student’s t test revealed no significant difference between the ratios of SNAP25:recombinant αSyn in the two variants. **p < 0.01, ****p < 0.0001 by one-way ANOVA (global) and Student’s t test (pairwise) that was applied between 3K and WT in both NTG and αSyn-KO synaptosomes; n ≥ 5 different experiments per condition.
Supplier Page from Abcam for Anti-SNAP25 antibody