Rabbit polyclonal anti nr2b antibody

Striatal tissues (three rats per group) were lysed in lysis buffer containing phe­nylmethanesulfonyl fluoride. The supernatant was collected after centrifugation at 12,000g for 10minutes at 4°C. After measurement of protein concentration using a bicinchoninic acid assay kit (Pierce, Rockford, IL, USA), samples contain­ing 20 μg protein were loaded onto 7.5% sodium dodecyl sulfate polyacrylamide gel, then transferred to PVDF after electrophoresis, blocked for 1h with 5% skim milk, and finally closed overnight with indicated antibodies, including [rabbit polyclonal anti-pNR2B-Tyr1472 antibody (1:1,000, Sigma, St. Louis, MO, U.S.A), rabbit polyclonal anti-NR2B antibody (1:1,000, Sigma, St. Louis, MO, U.S.A), mouse monoclonal anti-Fyn antibody (1:1,000, Upstate, Charlottesville, VA), or β-actin (1:1,000, abcam LTD, Cambridge, UK). (4°C, shaker)], the PVDF were washed 3 times in TBST (TBS with 0.05% v/v Tween-20) at room temperature and then incubated with horseradish peroxidase-conjugated secondary antibody diluted in TBST (1:2,000) for 1 h at room temperature followed by washing, signal detection was performed with an enhanced chemiluminiscence kit, and β-actin as an internal reference, finally analysis of the integral value of the optical density using image analysis software.