Active β catenin

Total proteins were isolated from pathological and control tissues homogenized in liquid nitrogen with a mortar and pestle, using RIPA lysis buffer (Cell Signaling Technology, Beverly, MA, USA). Proteins were quantified by Bradford Assay (Thermo Fisher Scientific, Waltham, CA, USA) and the protein lysates were subjected to electrophoresis, followed by immunoblotting. Then, 30 µg of total proteins was incubated with SDS-PAGE sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% beta-mercaptoethanol, and 0.004% bromophenol blue) at 100 °C for 5 min. Tris-glycine SDS running buffer (25 mM Tris, 250 mM glycine, 0.1% SDS) was used for electrophoresis. Proteins were separated for 60′ at 100 V in an 8% Tris/glyicine acrylamide gel (Mini PROTEAN electrophoresis cell). After electrophoresis, proteins were transferred onto the Immobilon-P PVDF membrane at 80 mA for 1 h by using Tris-glycine SDS (48 mM Tris, 39 mM glycine, 0.037% SDS) transfer buffer with 20% methanol in a Mini Trans-Blot Electrophoretic Transfer Cell. The blotted PVDF membranes were directly blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 h and incubated overnight 4 °C with primary antibodies diluted in TBST with 5% BSA. Primary antibodies were Active-β-Catenin (1:1000) (Cell Signaling Technology), APC (1:1000) (Merck-Millipore, Burlington, MA, USA), and β-actin (1:8000) (Sigma-Aldrich, St. Louis, MI, USA), used as a protein loading control. The blotted membranes were washed thoroughly with TBST before incubation with diluted (1:10,000) HRP-conjugated anti-rabbit or anti-mouse (Bethyl Laboratories, Montgomery, TX, USA). The immune complexes were visualized using the ECL western blot detection system (EuroClone) by using AllianceLD2 hardware (UVItec Limited, Cambridge, UK).

Western blotting was performed as previously described [31 (link)]. Aliquots of 40 μg of protein lysate were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Membranes were incubated with DACT2 (TA306668, Origene), active β-catenin (#4270; Cell Signaling Technology), total β-catenin (#9562; Cell Signaling Technology), MMP9 (ab76003, Abcam), MMP2 (ab86607, Abcam), c-Myc (#13987, Cell Signaling Technology), Cyclin D1(sc-450), p-GSK3β (sc-373800), Cdc25c (sc-13138), Cdc2 (sc-54), p-Cdc2 (pY15.44) (sc-136014), β-actin (sc-8432) (all from Santa Cruz Biotechnology, CA, USA), or CyclinB1 (ab32053, Abcam) primary antibodies. Proteins were visualized using an Immobilon Western Chemiluminescent HRP Substrate kit (Millipore Corporation, Billerica, MA, USA).