Brush Border Membrane Protein Analysis

Brush border membrane (BBM) for Western blot analysis was prepared from IEC-18 cells using a protocol that was described before [37 (link)]. Protein extracts from the BBM and total cell lysate of IEC-18 cells were prepared and analyzed by Western blot using previously used standard protocols [25 (link)]. In this study, rat ASCT1 and rat B0AT1 specific antibodies and secondary antibodies coupled to horseradish peroxidase, all obtained from Abcam (Abcam PLC, Cambridge, MA, USA), were used to detect the respective proteins. Ezrin was used as the loading control in all the Western blot experiments. Immunoprecipitation with rat B0AT1-specific antibody and Western blot with anti-3-nitrotyrosine antibody was done to analyze nitrosylation levels of B0AT1 protein from BBM. The protein density of the specific proteins was quantitated with a densitometric scanner FluorChemTM instrument (Alpha Innotech, San Leandro, CA, USA). Real-time quantitative PCR was performed with rat ASCT1-specific primers and probe (ThermoFisher Scientific Inc., Waltham, MA, USA). ASCT1 gene expression was standardized against β-actin expression for each of the experimental conditions.