Antibodies for fibronectin

The eyecups were lysed in radioimmunoprecipitation assay buffer. An equal amount of total proteins was subjected to electrophoresis on 10% SDS-PAGE and then electrophoretically transferred to a polyvinylidene fluoride film. The member was blocked with 10% non-fat milk for 2 h at room temperature (RT) and incubated with primary antibody overnight at 4°C. After several washes, the membrane was then incubated with secondary antibody for 1 h at RT. The signal was developed with an enhanced chemiluminescence reagent kit (NCM Biotech, Newport, RI, United States). The bands were quantified with an ImageJ density analyzer and normalized to GAPDH levels. Antibodies for vimentin, collagen-1, alpha-smooth muscle actin (α-SMA), and CTGF were purchased from Abcam (Cambridge, United Kingdom). Antibodies for fibronectin, Smad2/3, and TGF-β receptor 2 (TGF-βR2) were obtained from Santa Cruz Biotechnology (Dallas, TX, United States). Antibodies for glycogen synthase kinase 3 beta (GSK3β), phosphorylated-GSK3β (p-GSK3β), phosphorylated-Smad2/3 (p-Smad2/3), anti-non–phosphorylated β-catenin (non–p-β-catenin), and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, United States).

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).