Monoclonal antibody against α tubulin

After treatments, BV2 cell cultures were lysed in Tris-buffered saline (TBS, pH 7.6) containing 10% glycerol, 1% Nonidet P-40, EDTA 1 mM, EGTA 1 mM plus a complete protease inhibitors cocktail (Roche Diagnostics; Mannheim, Germany). The cell lysates were mixed with 5x Laemmli sample buffer and boiled for 5 min. Equal amounts of protein (30 µg) were resolved on 10% SDS-PAGE and electroblotted for 70 min at 90 V and 4°C to nitrocellulose (Amersham Biosciences; Amersham, UK). The membranes were blocked for 1 h at RT in 5% (w/v) dry skim milk (Sveltesse, Nestlé; Barcelona, Spain) diluted in TBS with 0.1% Tween® 20 (TBST) and the membranes were then probed overnight at 4°C with the antibody against iNOS diluted in 5% milk-TBST (1∶1,000; BD Pharmingen; San Diego, CA, USA). After extensive washing with 5% milk-TBST and antibody binding was detected for 1 h at room temperature with a horseradish peroxidase-conjugated anti-goat secondary antibody (1∶4,000; Bio-Rad; Hercules, CA, USA), which was visualized after rinsing enhanced chemiluminescence (Amersham Biosciences; Amersham, UK). The blots were stripped in 62.5 mM Tris-HCl, pH 6.8, containing 2% SDS and 0.7% β-mercaptoethanol and the were reprobed with a monoclonal antibody against α-Tubulin (1∶10,000; Sigma; Madrid, Spain).