Western Blot Analysis of Thyroid Proteins

Preparation of whole-cell lysates from thyroid glands has been described previously [50 (link)]. The protein sample (30 μg) was loaded and separated by SDS-PAGE. After electrophoresis, the protein was electrotransferred to a poly vinylidenedifluoride membrane (Immobilon-P; Millipore Corp., Billeria, MA, USA). The antibodies phosphorylated STAT3 (1:500 dilution), total-STAT3 (1:1,000 dilution), p-ERK (1:500 dilution), total-ERK (1:1,000 dilution), vimentin (1:1,000 dilution), and GAPDH (1:1,000 dilution) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies for fibronectin (1:200 dilution), integrin α6 (1:200 dilution), integrin β1 (1:200 dilution), integrin and β3 (1:200 dilution) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The blots were stripped with Re-Blot Plus (Millipore, Billeria, MA, USA) and reprobed with rabbit polyclonal antibodies to GAPDH. Band intensities were quantified by using NIH IMAGE software (Image J 1.47).