Stat3 antibody

Cells were lysed using the RIPA Lysis and Extraction Buffer (Life Technologies, Grand Island, NY) supplemented with protease inhibitors (Roche, Indianapolis, IN). The total protein was quantified with a Pierce BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s protocol. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking in phosphate-buffered saline/Tween-20 containing 5% non-fat milk at room temperature for 1 h, the membrane was incubated with primary antibody (Cell Signaling Technology, Danvers, MA) at 4 °C overnight using human IL-6R antibody (1:2000, #12786), JAK3 antibody (1:2000, #8863), STAT3 antibody (1:2000, #4904), pSTAT3 antibody (1:1000, #9145), EGFR antibody (1:2000, #4267).Then, the membrane was incubated with goat anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase (1:5000; Santa Cruz Biotechnology, Dallas, Texas). GAPDH (#2118) was used as a loading control. Proteins were visualized with LumiGLOchemiluminescent substrate (Cell Signaling Technology, Danvers, MA).

H-Ras MCF10A cells were treated with vehicle, curcumin or tetrahydrocurcumin for 12 h and digested with lysis buffer. Protein A/G agarose beads were washed with the same lysis buffer 5 times. The cell lysate of each group was immunoprecipitated with protein A/G agarose beads and STAT3 antibody (Santa Cruz Biotech) at 4  $\mathrm{°}\mathrm{C}$ overnight using a rotator. PC-3 cells were transiently co-transfected with Myc-tagged STAT3 and HA-tagged STAT3 vectors. Cells were then treated with DMSO, curcumin or tetrahydrocurcumin for 12 h. Myc-tagged STAT3 was immunoprecipitated with A/G agarose beads and Myc-tag antibody and protein was detected by HA-tag antibody.