Dynabeads protein

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.