Image quant analysis software

The KKU-213 cell line was lysed with radioimmuno-precipitation assay (RIPA) buffer containing protease inhibitor cocktails, 0.5 M NaF, 0.2 M NaVO₄, 1 M Tris-HCl pH 7.5, 0.5 M EDTA, 2.5 M NaCl, 10% (v/v) NP-40, 10% (w/v) SDS, Triton X-100, and deionized water. A protein assay was performed using bicinchoninic acid (BCA; Thermo ScientificTM, Rockford, IL, USA). A total of 20 µg of liver homogenate was fractionated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Whatman, Dassel, Germany). The membranes were incubated overnight at 4 °C with the primary antibody, followed by incubation with the appropriate secondary antibody at room temperature for 1 h in Enhanced Chemiluminescence Plus solution (GE Healthcare, Buckinghamshire, UK). The band intensity was quantified with Image Quant Imager and ImageQuant analysis software (GE Healthcare, Uppsala, Sweden). The intensity of the bands was estimated by ImageJ software (NIH, Bethesda, MD, USA). The membranes were also stained for β-actin as an internal control of protein loading.