Western blotting

Viable CD4 T cells were purified on a Ficoll gradient and treated with lysis buffer (RIPA buffer; 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS) containing protease inhibitor mixture (Roche), 1 mM NaF (Sigma-Aldrich) and 1 mM NA₃VO₄ (Sigma-Aldrich). Protein samples were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore). Primary antibody directed against STAT3, phospho-STAT3 (Cell signaling Technology), STAT4 (Santa Cruz) and phospho-STAT4 (Santa Cruz) and then HRP-cojugated Donkey anti-rabbit antibody (Affinity Bioreagents) were used to detect target protein by ECL detection kit (GE Healthcare).