STAT3 activation assay

The degree of STAT3 activation was determined by measuring the increased expression of phosphorylated STAT3 (pSTAT3) with Western blot analysis, as previously described¹⁵. Briefly, the solubilized cells 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 exposed to 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 exposed to anti-STAT3 antibody (sc-8019; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and visualized using a horseradish peroxidase-conjugated anti-mouse IgG antibody with ECL Western blotting detection reagent. The membranes were re-blotted with an anti-β-actin antibody (C4) (sc-47778; Santa Cruz Biotechnology, Inc.) as an internal calibration control.