Hybond membrane

Cells were harvested and lysed using ice-cold RIPA lysis buffer. All denatured protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Hybond membranes (Amersham, Munich, Germany). Following blocking for 2 h in 5% fat-free milk, the membranes were incubated with primary antibodies against GSK-3β, NF-kB, Cyclin D1, MMP9, and P21 (1:500, Proteintech, Proteintech Group, USA). Blots were washed with TBST and then incubated with secondary antibodies (1:5000). Bands were visualized using an enhanced chemiluminescence (ECL) system according to the manufacturer's protocol (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anti-GAPDH (1:2000) was set as the internal control.

Protein assays were performed according to the Bradford method using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% acrylamide gels, and then transferred to Hybond™ membranes (Amersham, Germany). The membranes were blocked overnight in 5% skimmed milk in Tris-buffered saline with Tween® 20 (TBST; 10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween® 20). For immunoblotting, the membranes were incubated for 1 h with the P-gp (P-glycoprotein, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GST-π (Abcam, Cambridge, UK) antibody, rinsed with TBST and incubated with anti-goat IgG antibodies conjugated to horseradish peroxidase (HRP; Dako, Carpinteria CA, USA) at a dilution of 1:1000. After applying electrochemiluminescent (ECL)-Plus detection reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the protein bands were visualized using an X-ray film (Fujifilm, Tokyo, Japan). The immunoblots were washed with western blotting (WB) stripping buffer (pH 2–3; Nacalai, Tokyo, Japan) and probed using a monoclonal antibody specific for GAPDH (1:1000; Proteintech Group, Chicago, USA).

A total of 30 μg of each cell or tissue lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were subsequently transferred to Hybond membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membranes were blocked and then incubated with the designated primary antibodies, and signals were detected using an ECL Western Blotting Kit (Amersham Pharmacia Biotech). Goat anti-BAMBI polyclonal antibody (R&D Systems, Minneapolis, MN, USA), anti-TGF-β1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) anti-β-catenin (BD Biosciences, Franklin Lakes, NJ, USA), anti-HBx (Chemicon International, Temecula, CA, USA) and β-actin (Sigma, Saint Louis, MO, USA) were used as the primary antibodies.

Protein assays were performed according to the Bradford method using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% acrylamide gels, and then transferred to Hybond-membranes (Amersham, Germany). The membranes were blocked overnight in 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST; 10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20). For immunoblotting, the membranes were incubated for 1 h with the primary antibody, rinsed with TBST and incubated with anti-rabbit, anti-mouse or anti-goat IgG antibodies conjugated to horseradish peroxidase (HRP; Dako, Carpinteria CA, USA) at a dilution of 1∶1000. After applying enhanced chemiluminescent (ECL)-Plus detection reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the protein bands were visualized using an X-ray film (Fujifilm, Tokyo, Japan). The immunoblots were washed with western blotting (WB) stripping buffer (pH 2–3; Nacalai, Tokyo, Japan) and probed using a monoclonal antibody specific for β-actin (1∶1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

The complete endometrial carcinoma proteome was extracted in radio-immunoprecipitation assay buffer, which prevented protease-mediated sample degradation. The protein concentration was determined for each sample. Then, 40 μg of the denatured proteome was resolved by 10 or 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then electrotransferred to Hybond membranes (Amersham, Munich, Germany). After the membranes were blocked with 5% fat-free milk at room temperature for 2 h, they were incubated with primary antibodies against RhoC (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), P70S6K, MMP2, and Bcl-xL (1:1000; Proteintech, Proteintech Group, USA) at 4 °C overnight. The membranes were washed three times with Tris-buffered saline containing Tween-20 (TBST), and then anti-rabbit or anti-goat secondary antibodies (1:5000) were added. After 2 h of incubation at room temperature, the protein bands were visualized by enhanced chemiluminescence according to the manufacturer’s instructions (Santa Cruz Biotechnology, Santa Cruz, CA, USA). β-actin (1:3000; Proteintech, Proteintech Group, USA) served as the loading control.

Cells were lysed with RIPA Lysis and Extraction Buffer (89901, Thermo Fisher, Waltham, MA, USA) including Halt^TM Protease Inhibitor Cocktail (78410, Thermo Fisher) and protein concentrations were determined using the Bradford method (Molecular Devices, Downingtown, PA, USA). Protein samples were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to Hybond membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Proteins were incubated with antibodies against GRIM19 (ab110240, Abcam, Cambridge, MA, USA), phospho-STAT3 (Tyr705; 9145, Cell Signaling, Danvers, MA, USA), phospho-STAT3 (Ser727; 9134, Cell Signaling), STAT3 (Cell 9139, Signaling), and β-actin (sc-47778, Santa Cruz Biotechnology). They were then detected using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL, USA) and HyperFilm (Agfa, Mortsel, Belgium), with β-actin used as a loading control.