Anti stat3 clone 124h6

48 hours after transfection, cells were lysed in a lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% Sodium Deoxycholate, 1 mM EDTA, 0.1% SDS, protease inhibitors). Proteins were separated by 4–15% SDS-PAGE and subsequently transferred to a nitrocellulose membrane (Fisher Scientific). The membranes were incubated overnight at 4 °C with the primary antibodies anti-tubulin (clone G-8, 1:1000, Santa Cruz Biotechnology, Dallas, TX, US), anti-phosphorylated STAT3 (Tyr705, clone D3A7, 1:2000, Cell Signaling) and anti-STAT3 (clone 124H6, 1:2000, Cell Signaling) in 5% bovine serum albumin in TBST buffer (25 mM Tris pH 7.4, 150 mM NaCl, 2.5 mM KCl, 0.05% Tween-20) at 4°C overnight, followed by a one-hour incubation with secondary horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG (Santa Cruz) and extensive washes with TBS containing 0.05% Tween-20. Phosphorylated STAT3, total STAT3, and tubulin proteins were detected by enhanced chemiluminescence (Pierce).