Mouse anti transferrin receptor

After boiling for 5 min, SDS-polyacrylamide gel electrophoresis (Bio-Rad, 456–8124) was used to separate 10 μl of the total and surface protein fractions for samples of GluN1 co-expressed with GluN2 A, while 5 μl of total protein and 30 μl of the surface protein fraction were separated for samples of GluN1 co-expressed with GluN2B. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes, and immunoblotted with following antibodies: mouse anti-GluN1 (BD Pharmingen, San Diego, CA, USA), mouse anti-transferrin receptor (Sigma, St Louis, MO, USA) and mouse anti-tubulin (Sigma). Tubulin was the internal control for total protein expression and monitors the quality of biotinylation experiment as tubulin is only present in the cytoplasmic fraction. Transferrin receptor was a control to show relatively equal surface biotin labeling. Bands were detected with film, and subsaturated bands were quantified with NIH ImageJ. Densitometry was used for chemiluminescence signal quantification, and the ratio of surface-to-total protein level of mutant was normalized to the wild type.
The statistical difference was determined by paired t-test. The number of independent experiments was represented by n. Samples sizes were determined by a priori power analysis for effect size = 2, power = 0.8 and α = 0.05.