Novel function of SART1 in HNF4α transcriptional regulation contributes to its anti-viral role during hepatitis B virus infection
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original data for Novel function of SART1 in HNF4α transcriptional regulation contributes to its anti-viral role during hepatitis B virus infection
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Immunofluorescence microscopy Cells grown on coverslips were fixed with 4% paraformaldehyde (Invitrogen, USA) for 10 min at room temperature and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 5 min on ice. Fixed cells were blocked in PBS containing 1% normal goat serum for 1 h at room temperature. After blocking, primary antibodies diluted with 1% blocking serum were added overnight at 4℃. After PBS washing, cells were incubated with the secondary antibody for 1 h in the dark at room temperature. Cells were mounted with Hoechst (Invitrogen, USA) for 5 min at room temperature and images were captured with an Olympus CKX53 FL Microscope (Olympus, Japan). Image processing was conducted by Image J software. Western blot Cell lysates were performed by lysing cells buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, protease inhibitor) for 30 min on ice. Cell lysates were spun down at 12,000 g for 10 min at 4℃ and the supernatants was collected, the protein concentration was determined using a Bradford Assay Kit (Bio-Rad, USA). Total proteins (30 μg) were separated by SDS-PAGE using Mini-PROTEAN Tetra (Bio-Rad, USA) and transferred into a PVDF membrane (Millipore, USA) using a Mini Trans-Blot system (Bio-Rad, USA). The membrane was then blocked with 2.5% nonfat milk for 1 h at room temperature and incubated with the primary antibody overnight at 4℃. Protein bands were detected using Immobilon Western chemiluminescent HRP substrate (Millipore, USA) by a Luminescent Image Analyzer (Syngene, England) and analyzed using Image J software.