Phospho tyrosine stat3

Cells were washed with cold PBS and lysed in a cold Radio ImmunoPrecipitation Assay (RIPA) buffer containing protease inhibitor (2 mM PhenylMethylSulphonylFluoried (PMSF), 10 μg/ml leupeptin and 2 mM EthyleneDiamineTetraAcetic acid (EDTA)). The lysates were collected and centrifuged for 20 min at 13,000 rpm at 4°C, and the supernatants were collected. Equal amounts of proteins from the supernatants were separated by SDS-PAGE and transferred on to nitrocellulose (NC) membrane. The membranes were blocked in a TBS-T containing 5% non-fat dried milk for at least one hour and subsequently incubated with specific primary antibodies overnight at 4°C. After washing with TBS-T for 30 min at room temperature, the membranes were further incubated with a HRP-conjugated secondary antibody for 1 h. After washing with TBS-T, the signals were detected using SuperSignal West Femto (Thermoscientific, USA). The following antibodies were used: phosphotyrosine STAT3, total STAT3, HA-tag, and ubiquitin (Cell signaling technology, USA), and active β-catenin, Myc-tag (Millipore), and total β-catenin (Santa Cruz Biotechnologies, USA) and α-tubulin (Thermoscientific).