Anti stat3 antibodies

Non-denatured cellular nuclear extracts were prepared using a NE-PER extraction kit (Thermo Scientific Pierce, Rockford, IL). Nuclear protein extracts were incubated with 4’ biotin-labeled DNA probes derived from the Wnt5a promoter sequence. Each probe was synthesized by Sigma-Genosys (The Woodlands, TX) and designed to include one of the above described 4 GAS-like elements in the Wnt5a promoter. The probes were incubated for 30 minutes on ice. Following incubation, the samples were separated on a 5% polyacrylamide gel, transferred onto a nylon membrane, and fixed on the membrane via ultraviolet cross-linking. The biotin-labeled probe was detected with streptavidin-horseradish peroxidase (Gel-Shift Kit; Panomics, Fremont, CA). The control consisted of a 7-fold excess of unlabeled cold probe. To test the effect of STAT3, anti-STAT3 antibodies (BD Biosciences) or mouse IgG1 (BD Biosciences) were added to the nuclear extracts, as previously described (10 (link)).

Western blot analysis was performed as described previously ^{10 (link)}. Briefly, cell lysates were assayed for their protein concentrations using the BCA protein assay reagent (Pierce Chemical, Rockford, IL). The lysates were mixed with 4x Laemmli sample buffer and then denatured by boiling for 5 minutes. Forty micrograms of lysates were separated using 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. The membranes were blocked with 5% dried milk dissolved in 50 mL of PBS. After blocking, the membrane was incubated with the following primary antibodies: anti-STAT3 antibodies (BD Biosciences, San Jose, CA), anti-Tyr705 STAT3 antibodies (Cell Signaling, Danvers, MA), anti–NF-κB p65 antibodies (Millipore, Charlottesville, VA), and mouse anti-human β-actin antibodies (Sigma-Aldrich, St. Louis, MO). After incubation with horseradish peroxidase–conjugated secondary antibodies (GE Healthcare, Buckinghamshire, UK) for 1 hour, the blots were visualized with an enhanced chemiluminescence detection system (GE Healthcare).