STAT3 Monoclonal Antibody (9D8)

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	-Mouse (1:5000)

Protocol tips
-Mouse (1:5000)

Publication protocol

The cells were rinsed with the lysis buffer (125 mmol/L Tris-HCl, 750 mmol/L NaCl, 1% v/v NP40, 10% v/v glycerol, 50 mmol/L MgCl2, 5 mmol/L EDTA, 25 mmol/L NaF, 1 mmol/L NaVO4, 10 μg/mL leupeptin, 10 μg/mL pepstatin, 10 μg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride; pH 7.5), sonicated and centrifuged at 13,000 x g for 10 min at 4°C. 20 μg of proteins from cell lysates were subjected to Western blotting and probed with the following antibodies against: IDO1 (rabbit polyclonal, diluted 1:2,000, AG-25A-0029, Adipogen, San Diego, CA); IDO2 (mouse monoclonal, diluted 1:500, SAB3701447, Sigma Chemical Co.); TDO (rabbit polyclonal, diluted 1:1,000, SAB2102400, Sigma Chemical Co.); phospho(Tyr 1022/1023)-JAK1 (rabbit polyclonal, diluted 1:1,000, #3331, Cell Signaling Technology, Danvers, MA); JAK1 (rabbit polyclonal, diluted 1:1,000, #3344, Cell Signaling Technology); phospho(Tyr701)-STAT1 (rabbit polyclonal, diluted 1:1,000, #9167, Cell Signaling Technology); STAT1 (mouse monoclonal, diluted 1:1,000, clone 15H3, Thermo Scientific, Rockford, IL); phospho(Tyr705)-STAT3 (rabbit polyclonal, diluted 1:2,000, #9145, Cell Signaling Technology); STAT3 (mouse monoclonal, diluted 1:5,000, clone 9D8, Thermo Scientific); Pgp (rabbit polyclonal, diluted 1:250, sc-8313, Santa Cruz Biotechnology Inc.); MRP1 (mouse monoclonal, diluted 1:100, ab32574, Abcam, Cambridge, UK); MRP2 (mouse monoclonal, diluted 1:100, ab3373, Abcam); MRP3 (goat polyclonal, diluted 1:250, sc-5776, Santa Cruz Biotechnology Inc.); MRP4 (goat polyclonal, diluted 1:250, ab77184, Abcam); MRP5 (goat polyclonal, diluted 1:250, sc-5781, Santa Cruz Biotechnology Inc.); BCRP (rabbit polyclonal, diluted 1:500, sc-25882, Santa Cruz Biotechnology Inc.); β-tubulin (mouse monoclonal, diluted 1:500, sc-5274, Santa Cruz Biotechnology Inc.), followed by a secondary peroxidase-conjugated antibody (Bio-Rad Laboratories). The proteins were detected by enhanced chemiluminescence (Bio-Rad Laboratories). Nuclear extracts were prepared with the Nuclear Extract Kit (Active Motif, Rixensart, Belgium); 10 μg of nuclear proteins were resolved by SDS-PAGE and probed with the following antibodies against: PIAS1 (rabbit monoclonal, diluted 1:1,000, ab109388, Abcam); PIAS3 (rabbit polyclonal, diluted 1:1,000, ab22856, Abcam); phospho(Tyr701)-STAT1; STAT1; phospho(Tyr705)-STAT3; STAT3; TATA-binding protein (TBP; rabbit polyclonal, diluted 1.500, sc-273, Santa Cruz Biotechnology Inc.). To exclude any cytosolic contamination of nuclear extracts, we verified that β-tubulin was undetectable in nuclear samples (not shown).

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