Image master id

Cells and resected tumor tissues were subjected to protein extraction using tissue protein reagents (T-PER, Pierce Biotechnology, Rockford, IL, USA). Proteins were separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were incubated with the following antibodies: cyclin D1, β-catenin, Src, phospho-Src, Akt, phospho-Akt, Smad2/3, phospho-Smad2/3, Thromboxane A2 Receptor (TXA2R) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Glucose Transporter-1 (GLUT1), PKM2, SLC1A5, SLC7A5, glutaminase, CD41, CD62P, CD45, SDF-1α, CXCR4 (Abcam, Cambridge, MA, USA), and Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) (R&D Systems, Minneapolis, MN, USA). The reacted proteins were further determined with horseradish peroxidase-labeled IgG, visualized using Enhanced Chemiluminescence (ECL) Western blotting reagents, and quantified by the optical densitometry (Image Master ID, Pharmacia Biotech, Upsalla, Sweden) of developed autoradiographs.

The excised liver tissues or white adipose tissues (including inguinal, periovarian, mesentery, and perirenal fat) were homogenized with tissue protein reagents (T-PER, Pierce Biotechnology, Rockford, IL, USA) and equally quantified before western blotting. Proteins were separated by electrophoresis with SDS-PAGE and then transferred to PVDF membranes. The membranes were sequentially incubated with 5% non-fat milk, recognizing antibodies, and horseradish peroxidase-labeled IgG. Antibodies recognizing phosphorylated Akt, Akt, phosphorylated AMP-activated Protein Kinase (AMPK), AMPK, phosphorylated Acetyl-CoA Carboxylase (ACC), ACC, uncoupling protein-1 (UCP-1), and β-actin were used (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Lastly, the blots were developed using enhanced chemiluminescence (ECL) substrates with autoradiograph, and quantified by optical densitometry (Image Master ID, Pharmacia Biotech, Upsalla, Sweden).