e., in aqueous medium at 37 °C. Uptake of DDV-Mas-7 by spinal cord neurons and release of dextran-Mas-7 from DDV-Mas-7 were determined by confocal

microscopy. Cells were exposed to DDV-Mas-7 (100 nM) in growth medium at 37 °C for 16 h, which was the optimum Selleckchem Belinostat time for the DDV uptake and separation (Zhang et al., 2009). Cells were subsequently washed and fixed overnight with 2% paraformaldehyde. The coverslips containing fixed cells were mounted between a glass slide and glass coverslip and fluorescent images were viewed on a Bio-Rad 2000 laser confocal microscope. For SNAP-25 immunoblotting, cells in each well were lysed in 1% SDS with 1 mM EDTA and 1 mM EGTA. The slurry was transferred to 1.5-ml microcentrifuge tubes, incubated in a 95 °C water bath for 5 min to inactivate proteases, and then stored at −80 °C. Immediately prior to use, samples

were thawed, mixed with equal volumes of Tris-Tricine sample buffer (Bio-Rad), heated at 95 °C for 4 min, and then separated by SDS-polyacrylamide gel electrophoresis. Equal quantities of protein were loaded onto 4–12% acrylamide gels. Proteins were separated using 0.1 M Tris-Tricine, pH 8.3, and then transferred to polyvinylidene difluoride membrane with a buffer containing 192 mM glycine, 25 mM Tris, pH 8.3, and 12% methanol. The membranes were soaked in 20 ml of 4% BSA in Tris-buffered saline-Tween 20 (TBST) buffer for 1 h at RT and next probed overnight with 2 ml of Goat anti-SNAP25 in the same buffer. After three 20-min washes with TBST, the membranes were exposed for 1 h to monkey Wilson disease protein anti-goat antibodies NVP-BGJ398 mw conjugated with horseradish peroxidase in 10 ml of blocking buffer. After washing three times with TBST, the membranes were developed with a chemiluminescent reagent (ECL). Same membrane was used for house-keeping protein analysis, i.e. after blotting with anti-SNAP25, the membranes were next used for anti-β-actin analysis. Scanned images of Western blots were produced and analyzed utilizing Bio-Rad quantity-one software. To determine whether Mas affects glycine exocytosis, 3[H]glycine release was measured in the same cell preparations of three-week

old mouse spinal cord neuronal cultures to examine a correlation between different mastoparans and 3[H]glycine exocytosis (Fig. 2). Stimulation with KCl (80 mM) released 3[H]glycine from untreated control cells, but the release was almost completely prevented (only 2% of untreated control) from BoNT/A (1 pM) poisoned cells. In these BoNT/A-treated cells, Mas (10 μM) or Mas-7 (10 μM) alone released about 30% and 50%, respectively, of the releasable pool of 3[H]glycine following 80 mM K+ stimulation. A combination of Mas plus 80 mM K+ increased 3[H]glycine release from BoNT/A treated cells about one and half-fold compared to that seen in untreated control cells (no BoNT/A); the release was even higher (two-fold over untreated control) with Mas-7 plus 80 mM K+.