*4.6. Western Blot Analysis*

Protein expression of LAT1, LAT2, p-4EBP1 (Thr-37/46), and p-S6k1 (Thr-389) was examined by Western blotting. An aliquot of 20 µg of total protein was subject to electrophoresis then transferred to nitrocellulose membrane. After blocking, the membranes were probed with anti-human antibody against LAT1, LAT2, p-4EBP1 (Thr-37/46), or p-S6k1 (Thr-389) and followed by corresponding biotinylate conjugated secondary antibodies. Antibodies against LAT1 and LAT2 were obtained from Santa Cruz Biotechnology; p-4EBP1 (Thr-37/46) and p-S6k1 (Thr-389) antibodies were purchased from Cell Signaling Technology (Danvers, MA). The bound antibody was visualized with an enhanced chemiluminescent detection kit (Thermo Scientific). β-actin expression was used as loading control for each sample. The band densities were scanned and analyzed by Alpha Imager software (Alpha Innotech Corporation, CA, USA).
