Hbp 023 9

Co-immunoprecipitation was performed as previously described [26 (link),44 (link)], with modifications. Briefly, human 293T cells were lysed for 30 min at 4 °C, using the lysis buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7, 1 mM EDTA, 0.5% IGEPAL, 1% Triton X-100), to which protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) was added; subsequently, the cell lysates were clarified. Immunoprecipitation was performed using Dynabeads™ Protein G (Catalogue number: 10004D) according to the manufacturer’s instructions. Mouse anti-HBV core (Thermo Fischer Scientific MA1-7606, Waltham, MA, USA) or mouse anti-MOV10 (Abcam ab 176687, Cambridge, MA, USA) antibodies were incubated with Dynabeads™ Protein for 24 h at 4 °C with rotation. Cell lysates were then mixed with the beads and incubated for 18–24 h at 4 °C with rotation. Where indicated, samples were incubated with RNase A (Qiagen, Germantown, MD, USA) (5 mg/mL) for 60–90 min at 4 °C prior to immunoprecipitation. Immunoprecipitates were then analyzed by SDS-PAGE followed by western blotting. For western blotting, membranes were probed with rabbit anti-HBV core antibody (Austral Biologicals HBP-023-9), 1:500 dilution, and rabbit anti-MOV10 antibody (Abcam, ab80613, Cambridge, MA, USA), 1:1000 dilution, overnight at 4 °C.

Cells were lysed using a radioimmunoprecipitation assay buffer (50 mM Tris/HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, pH 7.5) to which protease inhibitor cocktail (Sigma-Aldrich) was added. The protein concentration was measured using Bradford assay (Bio-Rad). A total of 10–20 µg protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride Immobilon-P membranes (MilliporeSigma, Burlington, MA, USA). Membranes were blocked with 5% non-fat milk in Tris Buffered Saline (TBS) with 0.1% Tween 20 (TBST). Membranes were probed with a rabbit anti-HBV core antibody (Austral Biologicals HBP-023-9, San Ramon, CA, USA) 1:500 dilution, rabbit anti-MOV10 antibody (Abcam, ab80613, Cambridge, MA, USA) 1:2,000 dilution, overnight at 4 °C, or mouse anti-GAPDH antibody (Santa Cruz Biotechnology sc-365062) 1:10,000 dilution at RT for 1 h, then anti-mouse (Sigma-Aldrich A9044, St. Louis, MO, USA) and anti-rabbit (Sigma-Aldrich A0545, St. Louis, MO, USA) horseradish peroxidase (HRP)-conjugated secondary antibodies were added at RT for 1 h. A Luminata Forte Western HRP substrate (MilliporeSigma, Burlington, MA, USA) was added to the membrane. Then, images were taken using a UVP BioSpectrum 815 imaging system (Analytic Jena, Upland, CA, USA).