Protein
AEBSF analysis using 2D-DIGE. Protein samples were homogenized with lysis buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 0.8 μM aprotinin, 15 μM pepstatin, 0.1 mM PMSF, 0.5 mM EDTA, 30 mM Tris–HCl, pH 8.5) and centrifuged at 13,000 rpm for 20 min at 4 °C. The supernatants were used as protein samples. The protein concentrations were determined with a protein assay reagent (Bio-Rad). The non-HCC and HCC samples (50 μg each) labeled with either Cy3 or Cy5 according to the manufacture’s manual were combined and separated on 2-DE gels together with the Cy2-labeled internal standard (IS), which was prepared by mixing equal amounts of all samples. Analytical 2-DE was performed as described previously [20] using Immobiline DryStrip (pH 3–10, 24 cm, GE Healthcare) in the first dimension and 12.5% SDS–polyacrylamide gels (24 × 20 cm) in the second dimension. Samples were run in triplicate to obtain statistically reasonable results. After scanning with a Typhoon 9410 scanner (GE Healthcare), gels were silver stained for protein identification. For protein identification, 400 μg of the IS sample was also separately run on a 2-DE gel and stained with SYPRO Ruby (Invitrogen). All analytical and preparative gel images were processed using ImageQuant (GE Healthcare) and the protein level analysis was done with the DeCyder software (GE Healthcare). To detect phosphoproteins, 400 μg of HCC and non-HCC samples were separately run on 2-DE gels and stained with ProQ Diamond (Invitrogen). After acquiring images, gels were counterstained with SYPRO Ruby to visualize total
proteins as described above.
