Anti 5 lo antibody

Tissue from the head of the pancreas obtained after control or NaT treatment as described previously was solubilized on ice in a lysis buffer consisting of NaCl (150 mmol/L), Tris, pH 7.5 (50 mmol/L), EDTA, pH 8.0 (10 mmol/L), dithiothreitol (1 mmol/L), Nonidet P-40 (1% v/v), sodium deoxycholate (0.5% w/v), sodium dodecyl sulfate (0.1% w/v), leupeptin (10 μg/mL), aprotinin (10 μg/mL), one tablet of protease inhibitors (cOmplete-Mini; Roche Diagnostics, Indianapolis, IN), and one tablet of phosphatase inhibitor (PhossSTOP; Roche Diagnostics) per 7 mL of solution. Supernates (10 μg protein) were loaded onto 4%–12% NuPage gels (Life Technologies), electrophoresed in 3-(N-morpholino)propanesulfonic acid buffer, and transferred onto polyvinylidene difluoride membranes (PerkinElmer, Waltham, MA). Blots were first reacted with a mouse monoclonal anti-5-LO antibody (BD Biosciences) at a dilution of 1/1000 and then with a rabbit polyclonal anti-α-tubulin antibody (GeneTex, Irvine, CA) at a dilution of 1/10,000. Bands were detected with the West Femto substrate (Thermo Scientific).