Rabbit polyclonal anti cbs

Cell pellets obtained from each experiment were mechanically homogenised in lysis buffer (RIPA supplemented with protease inhibitors cocktail) and total amount of protein was determined by using Bradford assay. Denatured proteins (60 µg) were separated on 10% sodium-dodecylsulfate polyacrylamide gels and transferred to polyvinylidene fluoride membrane (PVDF). Membranes were blocked in phosphate-buffered saline containing 0.1% v/v Tween-20 (PBST) and 3% w/v non-fat dry milk for 30 min, followed by overnight incubation at 4 °C with primary antibodies: mouse monoclonal anti-iNOS (1:500, BD-Pharmaingen), mouse monoclonal anti-COX-2 (1:2000, SantaCruz Biotechnologies), mouse monoclonal anti-CSE (1:1000, Proteintech), rabbit polyclonal anti-CBS (1:1000; Santa Cruz Biotechnologies), rabbit polyclonal anti-3MST (1:500, Novus Biologicals)⁵⁰ or mouse monoclonal anti-tubulin (1:5000, Sigma-Aldrich). Membranes were extensively washed in PBST prior to incubation with horseradish-peroxidase conjugated secondary antibody for 2 h at room temperature. Following incubation, membranes were washed and chemiluminescence was detected by using ImageQuant-400 (GE-Healthcare). The target protein band intensity was normalised against housekeeping protein α-tubulin.