Igfii

We used the following monoclonal and polyclonal capture and detection antibodies: PAPP-A (capture, Hytest, Turku, Finland; detection, R&D Systems, Minneapolis, MN, USA), fβ-hCG (capture, Acris Antibodies GmbH, Herford, Germany; detection, Hytest), AFP (both from Hytest), ANGPTL3 (both from R&D Systems), EGF (both from R&D Systems), IGFII (capture, Abcam, Cambridge, UK; detection, R&D Systems), SOD1 (capture, R&D Systems; detection, Hytest), and IgG (H + L) (Invitrogen, Breda, The Netherlands). Note that IgG was added for quality control purposes. Standards for PAPP-A and fβ-hCG were obtained from the AutoDELFIA kits. These standards were calibrated against the WHO International Reference Preparation (PAPP-A: 78/610 for SP1, fβ-hCG: 75/551). In addition to serum samples from the serum bank, the reference serum was used to correct for interarray variation.

Total protein from the human HCC cell lines was extracted using RIPA buffer (Pierce, Rockford, IL, USA). Equal amounts of protein for each sample were subjected to SDS-PAGE followed by transfer of protein to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were first incubated with antibodies against IGF-II (Abcam, Cambridge, UK), Ago1 (Millipore, Billerica, MA, USA), Ago2 (Millipore), RNAP II (Millipore), α-tubulin (Abcam), Topoisomerase I (Abcam), and GAPDH (Abcam), then followed by incubation with HRP-conjugated secondary antibodies. GAPDH (Abcam) was served as a loading control. The results were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA). Bands were quantified with Image-Pro Plus software (Media Cybernetics, USA). Each experiment was repeated thrice and all reactions were carried out in triplicate.