Western Blot Analysis of Caco-2BBE Cell Proteins

Caco-2BBE cells were washed with sterile phosphate-buffered saline and treated with RIPA buffer containing proteinase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Cell lysates were prepared and subjected to western blotting as previously described (Moon et al. 2022 (link); Um et al. 2023 (link)) using antibodies against ChREBP (NB400-13; Novus Biologicals, Centennial, CO, USA), GLUT5 (27571-1-AP; Proteintech, Rosemont, IL, USA), GAPDH (MAB374; Millipore, St. Louis, MO, USA), and TATA-binding protein (TBP) (ab63766; Abcam, Cambridge, UK). To detect nuclear ChREBP, nuclear lysates of Caco-2BBE cells were obtained using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents Kit (78833; Thermo Fisher Scientific, Carlsbad, CA, USA), according to the manufacturer’s instructions. Immunoreactive proteins were detected using an ECL kit (Thermo Fisher Scientific, Carlsbad, CA, USA), and the images were processed using Adobe Photoshop (Adobe, San Jose, CA, USA). Immunoreactive proteins were visualized using Amersham ImageQuant 800 (Marlborough, MA, USA). Protein bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).