Ab39716

Western blotting and immunohistochemistry were performed according to standard procedures^{50 (link)}. The following antibodies were used: anti-HBX (ab39716, Abcam, Cambridge, MA, USA), anti-P-CHK2 (2197S, Cell Signaling Technology, Danvers, MA, USA), anti-CHK2 (ab47433, Abcam), anti-ATM (ab199726, Abcam), anti-P21 (ab7960, Abcam), anti-P53 (BS1913, Bioworld, Minnesota, USA), anti-γH2AX (ab26350, Abcam), anti-P-CHK1 (2348S, Cell Signaling Technology), anti-CHK1 (10362-1-AP, Proteintech), anti-CDK2 (SC-6248, Santa Cruz Biotechnology, CA, USA), anti-Cyclin D1 (SC-718, Santa Cruz Biotechnology), anti-GAPDH (SC-47724, Santa Cruz Biotechnology), HRP-Ms (#7074, Cell Signaling Technology), and HRP-Rb (#7076, Cell Signaling Technology).

Fluorescent IHC staining was performed as described^{23 (link)}. Briefly, the frozen liver tissues from each treatment group of mice were sectioned and incubated with the primary antibodies anti-PPARγ (2435; Cell Signaling Technology), anti-HBsAg (1811; ViroStat, Portland, ME, USA), anti-HBx (ab39716; Abcam), or anti-CD45 (ab10558; Abcam), followed by the secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit IgG (ab150077; Abcam). Anti-HBsAg was used to detect pre-S2 mutant. DAPI (4’, 6-diamidino-2-phenylindole; Invitrogen) was used to stain the nuclei. For quantification, five independent microscopic fields (original magnification, ×40) with the most abundant nuclear PPARγ-, CD45-, pre-S2 mutant-, or HBx-positive cells in liver tissues of each mouse were selected. The total number of nuclear PPARγ-, CD45-, pre-S2 mutant-, or HBx-positive cells in the five selected fields of each mouse was counted manually and further calculated as the number of nuclear PPARγ-, CD45-, pre-S2 mutant-, or HBx-positive cells per field for statistical analysis.

The total protein was extracted from the biopsy tissue of the rat liver using the RIPA Lysis Buffer (Beyotime, P0013B, CN) according to the manufacturer’s instructions. The protein lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Epizyme Biomedical Technology, PG112, CN) and then electrophoretically transferred onto the polyvinylidene fluoride membranes (Epizyme Biomedical Technology, WJ001, CN). The primary antibodies we used were rabbit anti-HBx (ABCAM, ab39716, 1:1000, United States); mouse anti-GAPDH (MBL, M171-3, 1:5000, JPN). The ImageJ software was used to analyze the integrated density of the protein bands.