Stat3y705

Western blotting was performed according to a standard protocol. The primary antibodies used for probing were against α-tubulin (32-2500, Invitrogen; 1:2000), phosphorylated Stat3^Y705 (9131S, Cell Signaling Technology; 1:1000), Stat3 (610190, BD Biosciences; 1:1000), phosphorylated β-Catenin^{Ser33/Ser37/Thr41} (9561S, Cell Signaling Technology; 1:1000) and β-catenin (610153, BD Biosciences; 1:1000).

Cell pellets were lysed with RIPA buffer (Sigma-Aldrich) following the manufacturer’s instruction. SDS–polyacrylamide gel electrophoresis was performed using 4–20% Criterion TGX Stain Free Protein Gel (Bio-Rad, Hercules, CA), and proteins were transferred to poly-vinylidene difluoride (PVDF) Immobilion-p membrane (Merck-Millipore, Billerica, MA, USA) using wet/tank Bio-Rad blotting system. Membranes were blocked in I-block 2% (Invitrogen, Waltham, MA, USA), incubated overnight at 4 °C with primary antibodies and 1 h with the HRP- conjugated secondary antibody (PerkinElmer, Waltham, MA, USA). Bands were detected using Invitrogen™ iBright™ FL1500 Imaging System. The following primary antibodies were used: JAK1 Y1022/1023 (Cell Signaling Technologies, #3331), JAK2 Y1007 (MBS128333, MyBioSource, San Diego, CA, USA), STAT6 Y641 (Millipore, 06-937), STAT3 Y705 (Cell Signaling Technologies, #9145), STAT5 Y694 (Cell Signaling Technologies, #9351), STAT6 (#11290, Becton Dickinson, Franklin Lakes, NJ, USA), STAT3 (79D7) (Cell Signaling Technologies, #4904), STAT5 (Becton Dickinson #611834), GAPDH (GTX 8627408, Genetex, Irvine, CA, USA).