Anti α tubulin monoclonal antibody dm1a

Sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by western blotting was used to measure steady-state expression of CYE-1. For each sample, 50-100 gravid adult animals were boiled in 2X loading buffer and proteins separated on 4–15% precast gradient sodium dodecyl sulfate polyacrylamide gel electrophoresisgels (BioRad). Samples were transferred to nitrocellulose membrane and probed using anti-CYE-1 antibodies (1:2000 dilution; Brodigan et al. 2003 (link)). Anti-α-tubulin monoclonal antibody (DM1A; Sigma-Aldrich) was used at 1:5000 dilution. Supersignal (Thermo Scientific) was used for developing anti-α-tubulin and anti-CYE-1 western blots. The relative CYE-1 protein levels were quantified from scanned films using ImageJ.

All culture media (except EBM-2 and EGM-2), L-cysteine, fibronectin, trypsin, penicillin, and streptomycin were from Invitrogen (Breda, the Netherlands). EBM-2 and EGM-2 were from Lonza (Verviers, Belgium). Epinephrine, thrombin, forskolin, 3-isobutyl-1-methylxanthine (IBMX), LY294002, Y27632, BAPTA-AM, Endothelial Cell Growth Supplement (ECGS), and anti-α-tubulin monoclonal antibody (DM1A) were from Sigma-Aldrich Chemie (Steinheim, Germany). S1P was from Avanti Polar Lipids (Alabaster, Alabama, USA). Anti-β-catenin rabbit polyclonal antibody (sc-7199) was from Santa Cruz Biotechnology (Santa Cruz, California, USA). Chemiluminescence blotting substrate and Complete Protease Inhibitor Cocktail Tablets were from Roche Diagnostics (Mannheim, Germany). All chemicals used were of analytical grade. Anti-VWF monoclonal antibody CLB-RAg20 has been described previously [29] (link). Enzyme-linked immunosorbent assays (ELISA) for VWF and VWF propeptide have been described previously [30] (link).