Anti rabbit or anti mouse igg conjugated to horse radish peroxidase

For western blotting analysis, cells (T98 G or H460 cell lines)) or tissues (H460 cells) were harvested and then washed with ice-cold phosphate-buffered saline (PBS) twice before the addition of RIPA lysis buffer containing protease and phosphatase inhibitors (Roche, Indianapolis, IN, USA). Protein concentration was quantified using the bicinchoninic acid protein assay. Equal amounts of protein (20 μg per lane) were loaded into each well and separated by 7.5 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer onto polyvinylidene difluoride membranes (Bio-Rad, Richmond, CA, USA). Membranes were blocked using 5% nonfat dry milk or 2% bovine serum albumin (in PBS with 0.05% tween-20. The blots were then incubated with primary antibodies (1:200–1:1000) overnight at 4 °C. Secondary antibody anti-rabbit or anti-mouse IgG conjugated to horse radish peroxidase (1:5000; Cell Signaling Technology) was incubated for 2 h at room temperature. Immunoblots were developed using the chemiluminescence detection system (Thermo Scientific, Waltham, MA, USA). Quantification of immunoblotting signal of respective proteins was performed using the UN-SCAN-IT Gel software (Silk Scientific, Inc., Orem, UT, USA).

Immunoblotting was performed as previously described [19 (link)]. Protein samples (50 μg) were denatured, subjected to PAGE, and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Membranes were incubated with primary antibodies to GluN1 (BD Biosciences, Cayey, Puerto Rico), GluN2A (Sigma), or GluN2B (BD Biosciences) at 4 °C overnight, and then anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (Cell Signaling Technology) for 2 h at room temperature. Chemiluminescence (Visualizer Millipore, Charlottesville, VA, USA) was captured and quantified using a Kodak Imagestation 4000 MM.