Fig. 4
From: Intracellular interleukin-32γ mediates antiviral activity of cytokines against hepatitis B virus
IL-32γ downregulates HNF1α and HNF4α and reduces their binding to enhancers. a Map of liver-enriched transcription factor binding to HBV enhancers. b–d Huh7 cells were co-transfected with the HBV 1.2 and IL-32γ vectors or were treated with cytokines after HBV 1.2 transfection. The levels of transcription factors were determined by semi-quantitative RT-PCR (b), real-time PCR (c), or western blot analysis (d. e, f) Effect of IL-32γ and cytokines on the expression of HNF1α and HNF4α was analyzed by confocal fluorescence microscopy. At 24 h after transfection or treatment, immunofluorescence assay was performed using indicated antibodies (magnification, ×400; scale bar, 50 μm.). g, h Chromatin immunoprecipitation (ChIP) assay. Control or IL-32γ vector was transfected into Huh7 cells and ChIP assay was performed using anti-HNF4α (g) or anti-HNF1α (h) antibody. The level of the IL-32γ protein was determined by western blotting. The regions for R1–R3 were shown in above diagram (a). i Electrophoretic mobility shift assay (EMSA). An aliquot of 2 µg nuclear extracts was used. A cold competitor (50-fold) was used as a negative control. The protein complex was confirmed by western blotting using anti-HNF4α antibody. Data (c) was obtained from three independent experiments (mean ± S.D.). p < 0.001, p < 0.01 by Student’s t-test
