Phospho β catenin ser45

The cells were lysed at 4°C in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 5 mM EDTA (pH 8.0), and 1 mM EGTA supplemented with protease and phosphatase inhibitors. After 20 min of lysis on ice, the cell debris was removed via microcentrifugation, followed by rapid freezing of the supernatants. The protein concentration was determined using the Bradford method. In our experiments, equivalent loads of 25–50 g of protein were electrophoresed using a SDS-polyacrylamide gel and then electrophoretically transferred from the gel to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk, the membrane was incubated in specific primary antibodies (NIFK: Abcam, ab13880, 1:1000; Ki-67: Dako, Code M7240, 1:500; CK1α: Abcam, ab88079, 1:1000; non-phospho-β-catenin: Cell Signaling, #8814, 1:1000; phospho-β-catenin (Ser45): Cell Signaling, #9564, 1:1000; RUNX1: Cell Signaling, #4336, 1:1000) overnight at 4°C and subsequently incubated in a corresponding horseradish peroxidase-conjugated secondary antibody for 1 hr. The membranes were visualized using the ECL-Plus detection kit (PerkinElmer Life Sciences, Boston, MA, USA).