Western Blotting of Thyroid Proteins

For Western blotting, total cellular proteins were prepared from the thyroid tissues by the method described previously [27 (link)]. Fifty micrograms of sample protein was separated with 12% SDS/PAGE, and transferred to polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% skimmed milk in NaCl/Tris-T (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, and 0.5% Tween-20) at 4 °C overnight. It was rinsed three times (10 min each) with NaCl/Tris-T, and this was followed by 3 h of incubation with the same first antibodies that were used in immunohistochemical staining in the appropriate concentrations (IL-6, 1:500; COX-2, 1:500; NF-κB/p65, 1:500; β-actin, 1:3000, and IkBα, 1:1000) and 1 h of incubation with horseradish peroxidase-conjugated anti-rat IgG (Zymed Laboratories, San Francisco, CA, USA). Immunolabeling was detected with an enhanced chemiluminescence system (Roche, Mannheim, Germany), and visualized with the UVP Bio-spectrum Imaging System (UVP, Upland, CA, USA). β-actin was used as the internal quantitative control in densitometry analyses.