Rabbit anti stat3 antibody

Western blot analysis was performed to measure the expression of SOCS3, STAT3 and phosphorylated-STAT3 (pSTAT3). Whole-cell protein extracts from cells were prepared using lysis buffer for 30 min on ice. Protein concentrations were determined using an assay kit (Bio-Rad, Hercules, CA). Then, 50 μg of protein lysates were loaded, separated by denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA). Membranes were incubated in blocking buffer (Tris-buffered saline containing 5% skim milk) for 1 h at 37°C, followed by hybridisation with a rabbit-anti-SOCS3 antibody, a rabbit-anti-STAT3 antibody, and a rabbit-anti-pSTAT3 (tyr-705) antibody (1:1000 dilution, Cell Signaling Technology, USA) or a rabbit-anti-β-actin antibody (1:100 dilution, Lab Vision, Fremont, CA) at 4°C overnight. Then, membranes were hybridised with a horseradish peroxidase-conjugated rabbit immunoglobulin G (1:5000 dilution, Santa Cruz Biotechnology) secondary antibody for 1 hour at room temperature. Protein bands were detected by chemiluminescence using a western blotting luminol reagent (Santa Cruz Biotechnology). Films were scanned using a Gel Doc™ EZ Imager (Bio-Rad) and the protein level was semi-quantified using the Quantity One 1D image analysis software 4.4.0 (Bio-Rad).