Western blotting
SAP expression was measured using serum samples obtained from both groups. Bradford assay was performed to measure and quantify protein levels in each sample, followed by protein analysis using Western blotting with anti-JNK (Cat# SC-571 Santa Cruz, Santa Cruz, CA, USA), anti-p-JNK (Cat# SC-571 Santa Cruz, Santa Cruz, CA, USA), and anti-Elk-1 antibodies (Cat# SC-355 Santa Cruz, Santa Cruz, CA, USA). First, the total protein concentration in each serum sample was quantified using the Bradford method. Accordingly, 20 μg of protein was analyzed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 120 volts for 1.5 h.Subsequently, proteins in the gel were transferred to a nitrocellulose membrane (Bio-Rad, CA, USA) at 120 Volts for 1 h. The membranes were blocked for 30 min at room temperature with 5% blocking buffer, which was prepared by dissolving 2.5 mg of nonfat milk in 50 ml of washing buffer comprising 10 mM (5 ml of 2M stock) Tris-HCl (pH 7.5), 100 mM (20 ml of 5M stock) NaCl, 1 ml of 0.1% Tween 20, and 976 ml distilled water. The membranes were probed with primary antibodies against SAP, p-SAP, and Elk-1 (all 1:1,000 dilution), followed by incubation at 4°C overnight. After washing with washing buffer, the membranes were incubated for 1 h with horseradish peroxidase-conjugated polyclonal goat anti-rabbit IgG (1:10,000 dilution). The membranes were re-washed with washing buffer and developed on an X-ray film with ECL reagents (enhanced chemiluminescence kit, Youngin Frontier, Seoul, Korea).