Fusion fx5 imaging system

Samples were mixed with 2× Laemmli buffer prior denaturation at 95°C for 5 min. Samples were then subjected to SDS-PAGE and immunoblotting. Reagents for electrophoresis were purchased from BioRad and polyvinylidene fluoride membranes were from Millipore. The following antibodies (1:1000 dilution) were used: rabbit monoclonal anti-CNTFRα, anti-LIFRβ and anti-gp130 antibodies (Santa Cruz Biotechnology), anti-P-STAT3 (STAT3 phosphorylated on Tyr705) and anti-P-ERK1/2 (ERK1/2 phosphorylated on Tyr202/Thy204) antibodies (Cell Signaling Technology), mouse monoclonal anti-β-tubulin (Sigma), anti-flotillin (BD Biosciences), anti-STAT3 (Santa Cruz Biotechnology) and anti-ERK1/2 antibodies (Cell Signaling Technology). Goat anti-rabbit IgG and horse anti-mouse IgG peroxidase-conjugated secondary antibodies (Cell Signaling Technology) were detected using Supersignal chemiluminescence (ECL kit; Millipore). Approximate molecular weights were estimated using PageRuler prestained Protein Ladder Plus (Euromedex). Densitometric analysis of immunoblot images was performed using the Fusion Fx5 Imaging System (Vilber Lourmat) and ImageJ software.