Anti axin2

Kidney tissues were crushed in liquid nitrogen. Then, tissue samples or cell pellets were lysed in NP 40 lysis buffer (50 mM Tris-Cl, pH 8.0. 100 mM NaCl, 5 mM MgCl 2, 0.5% (v/v) Nonidet P-40) on ice for 30 min. The protein concentration was determined using a BCA Protein Assay Kit (Bio-Rad, CA, USA) following the manufacturer's procedure. Samples were run on a 10% SDS-polyacrylamide gel, transferred to an nitrocellulose membrane (Millipore) and immunoblotted with anti-collagen I, IV, anti MMP2, anti-a-SMA, anti-FN, anti-β-catenin, anti-axin2, anti-GSK-3β, anti-akt, anti-JNK, anti-c-jun, anti-c-fos and anti-P-Smad2,3 (Santa Cruz Biotechnology, CA) at 1∶1000 and anti-actin (Santa Cruz Biotechnology, CA) at 1∶5000. Rabbit anti-PA200 serum was diluted 1∶500 into phosphate-buffered saline–0.05% Tween (PBST)–1% ovalbumin. Antigen-antibody complexes were visualized with fluorescent labeled secondary antibodies (Sigma-Aldrich) diluted 1∶5,000 into PBST–1% ovalbumin and Fluorescent Western Blot Imaging Systems (Amersham).

Total protein extraction from tumor tissues using RIPA buffer (Cell Signaling Technology, Inc. Danvers, MA) and western blotting were performed as described previously studies [18 (link), 19 (link)]. The following primary antibodies were used: anti-Wnt-1, anti-β-catenin, anti-c-myc, anti-cyclin D1, anti-MMP-7, anti-axin-2, anti-APC, anti-c-jun, or anti-β-actin antibodies (Santa Cruz Biotechnology, Inc.). The secondary antibodies were corresponding to the type of primary antibody using anti-mouse or anti-rabbit IgG horseradish peroxidase (HRP)-conjugate (1:1,000 dilution, Cell Signaling Technology, Inc.). Protein bands were visualized by the Enhanced Chemiluminescence kit. The quantification of protein bands were measured by FluorChem Imaging Systems (Alpha Innotech). The data were standardized by β-actin.