Anti stat3 antibody 124h6

STAT3 activation was assessed by measuring the increased expression of pSTAT3 by western blotting, as previously described [37 (link)]. Solubilized SBC-3 cells and macrophages were run on a 10% SDS–polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) transfer membrane (Millipore, Bedford, MA, USA). To detect pSTAT3, the membranes were incubated with an anti-pSTAT3 antibody (D3A7; Cell Signaling Technology Japan, Tokyo, Japan) and visualized using a horseradish peroxidase-conjugated anti-rabbit IgG antibody with ECL western blotting detection reagent (GE Healthcare Life Sciences, Piscataway, NJ, USA). To detect STAT3, the membranes were incubated with an anti-STAT3 antibody (124H6; Cell Signaling Technology Japan, Tokyo, Japan) and visualized using a horseradish peroxidase-conjugated anti-mouse IgG antibody with an ECL western blotting detection reagent. The membranes were re-blotted with anti-β-actin antibody (C4) (sc-47778; Santa Cruz Biotechnology, Inc.) as an internal calibration control. Quantification of the western blots was performed using ImageJ and the Amersham Imager 680 analysis software.

We used a chromatin immunoprecipitation assay kit (Millipore, USA). In brief, cells were fixed with 1% formaldehyde (Beyotime Biotechnology, China) and harvested in SDS lysis buffer. DNA was sheared to fragments about 200–1000 bp by repeated sonication. Lysates containing soluble chromatin were precipitated overnight with 2 μg of anti-Stat3 antibody (124H6) (Cell Signaling Technology, USA) antibody or mouse IgG (#ab172730, Abcam). Protein A/G agarose was then added for additional 4 h. Protein-DNA crosslinks were reversed by treatment with proteinase K for 2 h at 45 °C. The DNA was subsequently purified, diluted and subjected in the quantitative real-time PCR reactions. The human XIAP and MCL1 gene promotor fragments were amplified. The promotor region of human GAPDH was used as a negative control. The primer sequences used are available upon request.