Anti rnf43

Protein extraction and western blotting were performed as previously described [16] . Tissue and cell lysates were prepared with lysis buffer. The protein content of the supernatant was measured using a standard method. The protein samples were loaded at 50μg/lane in 12% sodium dodecylsulfate (SDS), separated by polyacrylamide gel electrophoresis (PAGE), and transferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). The membranes were blocked with 5% non-fat milk in TBST (50 mmol/L Tris-HCl [pH 7.6], 150 mmol/L NaCl, 0.1% Tween 20) for 1 h at room temperature and then incubated with primary antibody (anti-RNF43, Abcam, USA, 1:200; GADPH, 1:1000, Santa Cruz Biotechnology) in blocking buffer at 4℃ overnight. After washing with TBST, the membranes were incubated with horseradish peroxidase-coupled goat anti-rabbit secondary antibody (1:10000; Santa Cruz Bio-technology) for 2 h at room temperature. Enhanced chemiluminescence was used for detection. The β-actin bands were used as the loading controls. The protein quantity was analyzed with Quantity-One v4.4 software (Bio-Rad, Hercules, CA, USA). The target protein expression was evaluated as the relative intensity ratio of the target protein to the loading control.