Supersignal west pico chemilumiscent substrate

Lysates containing 100 μg protein were mixed with loading buffer with 5% β-mercaptoethanol and heated for 5 min at 100°C. The protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes by semi-dry blotting. SDS-PAGE was performed via standard procedures according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). Membranes were incubated in blocking buffer [Tris-buffered saline (TBS), 0.1% Tween 20 and 5% low-fat dry milk] for 1 h at room temperature, followed by hybridization with anti-p-STAT3 (tyr-705) antibody (1:1,000 dilution; Cell Signaling Technology, Inc.), anti-STAT3 antibody (1:1,000 dilution; Cell Signaling Technology, Inc.) and anti-β-actin antibody (internal control; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight. Following three washes in TBS/0.1% Tween 20, the membranes underwent hybridization with a horseradish peroxidase-conjugated secondary antibody rabbit IgG (1:5,000 dilution; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Following extensive washing in TBS/0.1% Tween 20, signals were detected by electrogenerated chemiluminescence techniques using SuperSignal West Pico Chemilumiscent Substrate (Thermo Fisher Scientific).