Western blot and immunoprecipitation

Protein samples, prepared as described,19 were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking with non-fat milk, blots were incubated with primary antibodies against ROR2, phosphotyrosine, phosphoserine, GluN1, GluN2A, GluN2B, GluN2B–Ser 1303, GluN2B–Ser 1323, GluN2B-Tyr1336, GluN2B-Tyr1472, and β-actin overnight at 4°C. Blots were extensively washed and further incubated with HRP-conjugated secondary antibody at room temperature for 2 h, with immune complexes detected by using chemiluminescence (Pierce, Dallas, TX, USA). The intensity of each band was determined using Image J software (National Institutes of Health, Bethesda, MA, USA). For immunoprecipitation, the solubilised protein samples were preincubated with protein G/A-agarose for 6 h at 4°C and then incubated with 50 ml of protein G/A-agarose precoupled to antibody against primary antibodies for at least 3 h. The mixtures were then washed, boiled, and subjected to western blot procedures.