Mouse monoclonal anti stx1a

The lysates were obtained from DIV13-16 high-density neuronal cultures cultivated in 35 mm culture dishes. The neurons were lysed in 200 μl lysis buffer containing 50 mm Tris/HCl, pH 7.9, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 1% Nonidet P-40, and 1 tablet of Complete Protease Inhibitor (Roche) for 30 min on ice. Equal amounts of solubilized proteins were loaded in 12% SDS-PAGE and subsequently transferred to nitrocellulose membranes. The membranes were subjected to the following primary antibodies overnight at 4°C according to the experiment: mouse monoclonal anti-β-tubulin III (1:5000; Sigma) or mouse monoclonal anti-actin (1:4000) as internal controls, mouse monoclonal anti-STX1A (1:10,000; Synaptic systems), mouse monoclonal anti-SNAP25 (1:10,000; Synaptic systems), and rabbit polyclonal anti-STX3 (1:1000; Synaptic systems). HRP-conjugated goat secondary antibodies (Jackson ImmunoResearch) were applied for 1 hr at room temperature and detected with ECL Plus Western Blotting Detection Reagents (GE Healthcare Biosciences) in Fusion FX7 image and analytics system (Vilber Lourmat).

Protein lysates from cortical mass cultures were prepared by lysing neurons in 200 μl lysis buffer containing 50 mm Tris/HCl, pH 7.9, 150 mm NaCl, 5 mm EDTA, 1% Triton X- 100, 0.5% sodium deoxycholate, 1% Nonidet P-40, and 1 tablet of cOmplete Protease Inhibitor (Roche) for 30 min on ice. Samples were boiled for 5 min at 95°C. Equal amounts of protein were loaded onto a 12% SDS-PAGE and run in electrophoresis buffer at 80 V for 30 min and then 120 mV for 1 hr. Proteins were transferred onto a nitrocellulose membrane at 50 mA O/N. The membranes were blocked in 5% milk for 1 hr at RT and incubated with the corresponding primary antibody diluted in PBS for 1 hr at RT. Membranes were incubated with mouse monoclonal anti-STX1A (1:10,000; Synaptic Systems), rabbit polyclonal anti-STX2 (1:10,000; Synaptic Systems), rabbit polyclonal anti-STX3A (1:10,000; Synaptic Systems), rabbit polyclonal anti-Munc18-1 (1:10,000; Sigma-Aldrich), and mouse monoclonal anti-βTubIII (1:10,000; Sigma) (as loading control). Secondary antibodies conjugated with HRP-conjugated goat secondary antibodies (1:10,000; Jackson ImmunoResearch) diluted in PBT were applied for 1 hr at RT. Membranes were then incubated with ECL Plus Western Blotting Detection Reagents (GE Healthcare Biosciences) and luminal signal was visualized and imaged in Fusion FX7 image and analytics system (Vilber Lourmat).

Antibodies were used at the following dilutions and obtained from the following sources: mouse monoclonal anti-Stx1A (1:1,000), rabbit polyclonal anti-Stx3 (1:250), and rabbit polyclonal anti-Stx4 (1:250; Synaptic Systems, Göttingen, Germany), mouse anti-NgCAM 8D9, which recognizes an extracellular NgCAM epitope (1:5 of hybridoma supernatant; Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti-c-myc 9E10 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), rat anti-c-myc JAC6 (1:1,000) and rabbit anti-GFP (1:2,000; Abcam, Cambridge, MA), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:500; Chemicon, Billerica, MA), mouse anti-MAP2 (1:500; Sigma-Aldrich, St. Louis, MO), and mouse anti-Tau (Tau-5, 1:1,000; Invitrogen, Grand Island, NY). Alexa Fluor-conjugated secondary antibodies that were highly cross absorbed against multiple species (1:300), and Alexa 488-conjugated rabbit polyclonal anti-GFP (1:1,000) were from Molecular Probes (Invitrogen). Cy3-conjugated secondary antibodies (1:300) were from Jackson ImmunoResearch Laboratories (West Grove, PA). For Western blotting, Alexa 680-conjugated secondary antibodies (1:5,000) were from Molecular Probes and IRDye 800nm-conjugated secondary antibodies (1:5,000) were from Rockland (Gilbertsville, PA).