Amido black staining solution for total proteins

Cellular and placental membrane protein expression of System L isoforms was determined by Western blot analysis, carried out as previously described [26 (link), 39 (link)]. Briefly, cell protein lysates were prepared in RIPA buffer and protease inhibitors and phosphatase inhibitor cocktail 1 and 2 (1:100, Sigma Aldrich) were added to cell lysates and MVM and BM vesicles. Proteins were separated by SDS-PAGE electrophoresis using Mini-Protean TGX precast gels (Bio-Rad, Hercules, CA) and transferred to PVDF membranes (35 V constant, overnight at 4 °C). Membranes were stained with Amido Black staining solution for total proteins (Sigma-Aldrich) according to the manufacturer’s instructions and then blocked in 5 % non-fat milk in Tris-buffered saline containing 0.1 % Tween (TBS-Tween) for 1 h at room temperature. After brief washing in TBS-Tween, membranes were incubated overnight with antibodies targeting LAT1 and LAT2 (2 μg/ml) or beta-actin (0.2 μg/ml; Sigma-Aldrich). After washing, the membranes were incubated with the appropriate peroxidase conjugated secondary antibody and visualised using ECL detection solution (Thermo Scientific). Densitometry analysis was performed with ImageJ software (National Institutes of Health, USA). Target protein expression was normalized to beta-actin expression.