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Plasmids. The HBV, HBx and P53 protein expression vectors pCMV-HBVa, pCMV-HBVb, pCMV-HBx, pCMV-wtp53 and PT7HBc were supplied by the Center for Human Virology of Thomas Jefferson University. pG13-CAT reporter plasmid, its inner control plasmid pSVβ-gal and p21-luc were kindly provided by Vogelstein [23].
Preparation of nuclear extracts. Nuclei protein extraction from hepatoma GDC0941 was performed by Dignam’s method [25] and [26].
Electrophoretic mobility shift assay and in situ ultraviolet cross-linking assay. Electrophoretic mobility shift assay (EMSA) and in situ ultraviolet cross-linking assay were performed using a DNA–protein binding detection kit (Gibco-BRL) according to the manufacture’s protocol, which was shown in Supplementary material 1 [27].
Electrophoretic mobility supershift assay (EMSSA). For monitoring P53 composition in electrophoretic mobility supershift assay (EMSSA), 6 μg nuclei extract of HepG2 cells was incubated with 3 μl anti-P53 monoclonal antibody DO-1 (Oncogene) at 4 °C for 30 min, then a labeled fragment of synthesized complementary oligonucleotides was added, and the mixture was performed without nuclei extract or with anti-Ras antibody in place of anti-P53 antibody. The gel was run as EMSA, and exposed to Kodak X-ray film overnight at ?70 °C.