Tissue lysates were prepared in RIPA buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 10% Glycerol, 1% Triton X-100, 20 mM Na4P2O7, 1 mM PMSF, 2 mM NaVO3, 1 mM NaF, 0.1% SDS, 0.5% deoxycholate ) with protease (Complete EDTA-free, Roche), and phosphatase inhibitors (PhosSTOP, Roche). Samples were resolved on a 4–12% Bis-Tris gel and transferred onto nitrocellulose membranes. Proteins were detected using the following antibodies: anti-β-klotho (0.1 µg/mL, R&D systems, AF2619), anti-phospho ERK1/2 (1∶1000, Cell Signaling, 9101), anti-ERK1/2 (1∶1000, Cell Signaling, 9102), anti-phospho FGFR1 (1∶1000 Novus Biologicals, NB110-62076), anti-FGFR1 (Abcam, ab10646), anti-phospho FRS2-α (Cell Signaling, 3861), anti-FRS2 (1∶200, Santa Cruz Biotechnology, sc-8318), anti-phospho SHP2 (1∶1000, Cell Signaling, 3751), anti-SHP2 (1∶1000, Cell Signaling, 3752), and anti-β-actin (1∶5000, Sigma-Aldrich, A2228). The bands were visualized by chemiluminescence (Supersignal West Dura substrate, Pierce).
