Stat6

Liver tissues and cellular proteins were extracted with ice-cold lysis buffer [1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10% glycerol, 137 mM sodium chloride, and 20 mM Tris (pH 7.4)]. Proteins (20 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride nitrocellulose membranes. Phospho-STAT1, STAT1, phospho-STAT6, STAT6, phospho-AMPK, AMPK, phospho-AKT, AKT, Bcl-2, Bcl-xl, and β-actin rabbit mAbs (Cell Signaling Technology, MA, USA) were used. HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology, MA, USA) was used as the secondary antibody.

The following chemicals were used: 3-methylcholanthrene (MCA), NaCl, NH₄Cl, NaHCO₃ Hank's BSS without Ca²⁺ and Mg²⁺, Trypsin-EDTA solution (T3924) and LPS from Sigma-Aldrich (Munich, Germany), Dulbecco's PBS (Gibco, Life Technologies, Carlsbad, CA, USA), Hank's buffer, Sybr Green from Bio-Rad (California, USA), Tris (Carl Roth) NFκB inhibitor #sc-3060 from Santa Cruz (Texas, USA), and GKT 137928 from Genkyotex (Switzerland). IL4, IL13, and IFNγ were purchased from PeproTech (NJ, USA). The following antibodies were used: anti-β-actin (AC-15) from Sigma-Aldrich (Munich, Germany), pSTAT6, STAT6, pSTAT1, and STAT1 from Cell Signaling (Danvers, MA, USA), and p65, β-tubulin, and topoisomerase from Santa Cruz (Texas, USA). YM1 was from Chemicon-Millipore (Darmstadt, Germany), and YY1 was from Bethyl Laboratories (Texas, USA).

Cells were harvested after 5 days of incubation at 37 °C in humidified incubator and were lysed in ice-cold radioimmunoprecipitation (RIPA) lysis buffer (Catalog No. 89901, Thermo Scientific, Rockford, USA) containing protease and phosphatase inhibitor cocktails (Roche Diagnostic, Germany). Protein concentrations were determined by BCA protein assay Kit (Catalog No. 23225, Thermo Scientific, Rockford, USA) as described by manufacturer instructions. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (PVDF). After blocking, membranes were incubated with primary antibodies such as p-p38 (9211S, Cell Signaling), p38 (9212S, Cell Signaling), P-JNK (9251S, Cell Signaling), JNK (9252S, Cell Signaling), P-JAK1 (3331S, Cell Signaling), JAK1 (3332S, Cell Signaling), P-JAK3 (5031S, Cell Signaling), JAK3 (8863S, Cell Signaling) P-STAT6 (9361S, Cell Signaling), STAT6 (9362S, Cell Signaling), p-NF-κBp65 (SC-136548, Santa Cruz), NF-κBp65 (SC-109, Santa Cruz) and GAPDH (SC-32233, Santa Cruz) at 1:1000 dilutions for overnight at 4 °C. Membranes were then washed and incubated with horseradish-peroxidase conjugated secondary antibodies (1:5000 dilutions) for 2 h at room temperature. The blots were then detected with western blot detection kit (WesternBrightTMECL, USA).

Phospho-specific antibodies to STAT1^Tyr701, STAT3^Tyr705, STAT5^Tyr694 and STAT6^Tyr641 were from Cell Signaling Technologies (Beverly, MA, USA). STAT1, STAT3, STAT5, STAT6, JAK1, JAK2, JAK3, PTPN6, Bcl-2, c-Myc, Mcl-1 and survivin antibodies were also from Cell Signaling Technologies. Actin antibody was purchased from Santa Cruz (Santa Cruz, CA, USA). Recombinant human IL-2, IL-6 and IL-10 were from Peprotech (Rocky Hill, NJ, USA). IFN-α was purchased from Sigma Aldrich (St. Louis, MO, USA).

Recombinant human IGF2 (rhIGF2) was from R&D Systems (Minneapolis, MN, USA). Rapamycin, Entinostat, SU6656, and WAY-600 were from Selleck Chemicals (Houston, TX, USA). Cantharidic acid, Cypermethrin and BN-82002 were from Merck (Darmstadt, Germany), Endothall, RK-682, Deltamethrin, RWJ-60475, Tyrphostin 8, CinnGel, and BML-260 were from Enzo Biochemicals (New York, NY, USA). Primary antibodies used in western blot assays: IRS1 (#2382, 1:1000), IGF-1R (#3018, 1:1000), p-IGF-1R(Tyr1131)/IR(Tyr1146) (#80732, 1:1000), Akt (#4691, 1:1000), p-Akt(Thr308) (#13038, 1:1000), p-Akt(Ser473) (#4060, 1:1000), S6K (#2708, 1:1000), p-S6K(Thr389) (#97596, 1:1000), FOXO3a (#12829, 1:1000), p-FOXO3a(Ser253) (#9466, 1:1000), STAT6 (#5397, 1:1000), p-STAT6(Tyr641) (#56554, 1:1000), Src (#2109, 1:1000), p-Src(Tyr416) (#59548, 1:1000), and HDAC1 (#34589, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies used in western blot assays: PPP3CB (#HPA008823, 1:1000), PPP3CA (#WH0005530M3, 1:1000), PPP3CC (#SAB1409493, 1:1000), PPP3R1 (#WH0005534M1, 1:1000), PPP3R2 (#SAB1406289, 1:1000), and β-actin (#A5316, 1:3000) were from Sigma (St. Louis, MO, USA). HRP-conjugated secondary antibodies (Goat anti-Rabbit IgG, #31460, 1:5000 and Goat anti-Mouse IgG, #31430, 1:5000) were from Thermo Scientific (Waltham, MA, USA).