Western Blotting of FGF Family Proteins

Lysates from sFBs were prepared using RIPA buffer (25 mM Tris–HCl [pH 7.6], 150 mM sodium chloride, 1% NP-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)) containing protease inhibitors (Roche, Basel, Switzerland) according to the standard method [13 (link)], followed by centrifugation at 14,000×g for 15 min at 4 °C, and the concentration of each sample using a Bio-Rad protein assay kit (Bio-Rad, Tokyo, Japan) with bovine serum albumin as a standard. The sample was electrophoresed in 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane and incubated with primary antibodies: FGF7 (1:1000; abcam, Tokyo, Japan), FGF9 (1:1000; abcam, Tokyo, Japan), and GAPDH (1:5000; Sigma-Aldrich, Tokyo, Japan). After washed by TBST (20 mM Tris–HCl, 150 mM NaCl, and 0.02% Tween-20, pH 7.4), the blots were incubated with secondary antibodies conjugated with horseradish peroxidase (1:4000, anti-rabbit; GE Healthcare, Tokyo, Japan) for 1 h at room temperature. The bands were detected using ECL-Plus Substrate (GE Healthcare, IL, USA) and exposed to Hyperfilm (GE Healthcare, IL, USA). The data was obtained from an experiment using samples from a single donor.