Immunoblot Analysis of ASCT2 Protein

Immunoblot analysis was performed as previously described (22 (link)). Briefly, 2 million cells were pelleted and lysed in RIPA lysis and extraction buffer (ThermoFisher Scientific). Protein extracts were separated by electrophoresis on 12% SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (EMD Millipore). Membranes were blocked in 5% nonfat milk in PBS buffer and incubated with the rabbit anti-ASCT2 antibody [1:1,000, 8057S, Cell Signaling Technology (CST)]. Detection of GAPDH was used as a protein loading control. Immunoreactive bands were detected with horseradish peroxidase (HRP) anti-rabbit antibodies using the ECL system. Analysis of immunoblotting was performed using the LI-COR Biosciences Odyssey Imaging System.