Anti n cadherin monoclonal antibody

Proteins were fractionated on an SDS-PAGE gel and transferred to an Immobilon-P PVDF membrane (0.45 μm, Millipore). After blocking, membranes were incubated with primary antibodies and then anti-rabbit or anti-mouse secondary antibodies were used for detection. Immunoreactive proteins were visualized using Western Lightening Plus-ECL (PerkinElmer, #NEL105001EA). The following primary antibodies were used: anti-β-actin monoclonal antibody (Sigma, #A5441); anti-USP5 monoclonal antibody (Santa Cruz, #sc-390,943), anti-β-catenin monoclonal antibody (BD Biosciences, #610,153), anti-Slug polyclonal antibody (Santa Cruz, #sc-10,436), anti-E-cadherin monoclonal antibody (BD Biosciences, #610,182), anti-N-cadherin monoclonal antibody (BD Biosciences, #610,921), anti-Vimentin monoclonal antibody (Cell Signaling Technology, #5741S), and anti-Ubiquitin monoclonal antibody (Santa Cruz, #sc-8017).

Biotinylated or immunoprecipitated proteins and cell lysates were separated using 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with the Laemmli buffer system and then transferred to nitrocellulose membranes. After blocking with 5% nonfat dried milk in PBS containing 0.05% Tween 20, the membranes were incubated with specific primary antibodies (final concentration 1 μg/ml) followed by appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (final concentration 1 μg/ml). Proteins were then detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) using a Luminoimage Analyzer LAS-3000 (FujiPhoto Film). The antibodies used in the immunoblotting (or immunoprecipitation) were as follows: HNK-1 monoclonal antibody (a hybridoma cell line was purchased from the American Type Culture Collection), anti-GluA1 polyclonal antibody (Enzo Life Sciences), anti-GluA2/3 polyclonal antibody (Enzo Life Sciences), anti-N-cadherin monoclonal antibody (BD Biosciences). Each immunoblotting experiment was replicated two or three times, and representative figures are shown for each experiment.