Horseradish peroxidase conjugated

Hippocampal neurons from Munc13-1/2 DKO expressing Munc13–1 full length, Munc13–1 V549E,L554E, Munc13–1 R750E,K752E and Munc13–1 N940W mutants were lysed after 15 DIV at 4°C with RIPA lysis buffer including protease inhibitor cocktail-complete mini (Roche Diagnostics, Berlin, Germany). Equal amounts of proteins from the lysates of the four different groups were mixed with Laemmli sample buffer containing 0.1 M DTT, and boiled 5 min at 99°C. Protein lysates were separated on SDS polyacrylamide gels (4–8%% SDS-PAGE) and transferred to a polyvinyl difluoride (PVDF) membrane. Membranes were blocked for 1 hr with 5% skim milk in TBST and incubated at 4°C over night with primary antibodies: anti-Flag M2 (F1804; Sigma-Aldrich), and anti-Living Colors GFP (632375; Clontech, Mountain View, CA). Secondary antibodies were horseradish peroxidase-conjugated (Jackson ImmunoResearch, West Grove, PA). The immunoreactive proteins were detected by ECL Plus Western Blotting Detection Reagents (GE Healthcare Biosciences, Pittsuburgh, PA) in a Fusion FX7 detection system (Vilber Lourmat, Eberhardzell, Germany). Data were collected from three separate Munc13-1/2 DKO cultures and analyzed offline using ImageJ.

Cell pellets were lysed in buffer containing 50 mM Tris pH 8.0, 5 mM EDTA, 0.5% NP-40, and 100 mM sodium chloride for 20 minutes at 4°C. Protein concentration was quantified using the Bradford Protein Assay (Bio-Rad). Membranes were blocked for one hour in 5% milk in TBS-T. The primary antibodies used were mouse monoclonal anti-FLAG M2 (Sigma; catalog number F1804) diluted 1:1000; rabbit polyclonal anti-C/EBPβ (Santa Cruz sc-150) diluted 1:2500; rabbit polyclonal anti-c-Myb rabbit polyclonal (Santa Cruz sc-517) diluted 1:250; and anti-γ-actin (Novus; catalog number NB600-533) diluted 1:5000. All secondary antibodies from the appropriate species were horseradish peroxidase–conjugated (The Jackson Laboratory) and diluted at 1:10,000. All antibodies were diluted in 2% BSA (Sigma-Aldrich) in TBS-T or 1% milk in TBS-T.

Tissues were homogenized on ice in the lysis buffer. After centrifugation and protein quantification, proteins were loaded onto SDS–PAGE gels and transferred onto nitrocellulose membranes. Membranes were incubated in 5% nonfat dry milk in TBST for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies. Rabbit anti-DSC2 and mouse anti-β-actin antibody were purchased from Abcam Inc (Cambridge, MA). Antibodies were detected using 1:10,000 horseradish peroxidase-conjugated, donkey anti-rabbit, and donkey anti-mouse IgG (Jackson ImmunoResearch, USA). The Western blot luminol reagent was used to visualize bands corresponding to each antibody.

Briefly, retinal lysates were centrifuged at 12,000 ×g for 10 min at 4°C. Ten micrograms of each sample was separated by SDS-PAGE and electrotransferred to PVDF membranes (Immobilon-P; Millipore, Billerica, MA, United States). The membranes were blocked with non-fat milk (5%) for 1 h at room temperature and incubated with the following primary antibodies overnight at 4°C: a mouse monoclonal antibody against GS (ab64613, 1:1000; Abcam, Cambridge, MA, United States) and a rabbit monoclonal antibody against EAAT2 (ab205248, 1:1000; Abcam, Cambridge, MA, United States). The membranes were incubated with horseradish peroxidase-conjugated AffiniPure goat anti-mouse IgG (H + L) (115-035-003, 1:5000; Jackson ImmunoResearch Laboratories, West Grove, PA, United States) and horseradish peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) (111-035-003, 1:5000; Jackson ImmunoResearch Laboratories, West Grove, PA, United States). The relative intensities of the protein bands were quantified by scanning densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, United States). GAPDH was used as an internal standard.

Vero cells were washed with PBS, resuspended in cold lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, phosphatase inhibitor mixture (Sigma, St. Louis, MO, USA), and 1% Triton X-100, pH 7.4), and incubated for 30 min on ice. After centrifugation (10,000× g for 30 min at 4 °C) the supernatants were collected and assayed to determine their protein concentration (Bradford method, Bio-Rad, Hercules, CA, USA). Equivalent amounts of proteins were separated with SDS-PAGE, and blotted onto nitrocellulose membranes for Western blot analysis. The membranes were blocked with 10% nonfat dry milk in TBS, 1% Tween-20 for 1 h at room temperature, and incubated with primary antibodies: goat polyclonal anti-HSV (AbD Serotec, Oxford, UK), mouse monoclonal anti-glycoprotein B (Santacruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-NOX4 (Santacruz Biotechnology, Dallas, TX, USA), or mouse monoclonal anti-tubulin (Sigma Aldrich, St. Louis, MO, USA) at a final concentration of 1 µg/mL. Secondary antibodies were horseradish peroxidase-conjugated (Jackson ImmunoResearch, West Grove, PA, USA). Blots were developed with the Pierce ECL Plus Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA) and subjected to densitometric scanning.

Hippocampal neurons from E18.5 Munc13-1/2 DKO at a density of 10.000 / cm² were plated into 6 well plates containing monolayer cultures of astrocytes. Neurons were lysed after 15 DIV at 4°C with 50 mM Tris·HCl, pH 7.9, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 250 μM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, and protease inhibitor cocktail-complete mini (Roche Diagnostics, Berlin, Germany). Lysates were mixed with Laemmli Buffer containing 0.3 mM DTT, and boiled 10 min at 95°C. 30 µg of protein lysates were used for the SDS-PAGE electrophoresis. After separation by SDS-PAGE proteins were transferred to a polyvinyl difluoride (PVDF) membrane. Membranes were blocked with 5% skim milk in TBST, and incubated at 4°C over night with primary antibodies: anti-Flag M2 (F1804; Sigma-Aldrich), and anti-Living Colors GFP (632375; Clontech; Mountain View, CA). Secondary antibodies were horseradish peroxidase-conjugated (Jackson ImmunoResearch; West Grove, PA). The immunoreactive proteins were detected by ECL Plus Western Blotting Detection Reagents (GE Healthcare Biosciences; Pittsburgh, PA) in a Fusion FX7 detection system (Vilber Lourmat, Eberhardzell, Germany).