Northern Blot and Western Blot Analyses

Northern blotting was performed as previously described (Sun et al., 2017). Briefly, total RNA samples extracted from cells transfected with HBV plasmids were separated on 1.2% agarose gel with 7% formaldehyde. The samples were transferred to a nylon membrane (Roche Diagnostics, Basel, Switzerland), followed by cross-linking to the membrane by ultraviolet light. The blot was prehybridized with DIG Easy Hybridization buffer (Roche Diagnostics) and hybridized with an HBV RNA probe (Supplementary Figure S1A) labeled with DIG-11-UTP using the DIG Northern Starter Kit (Roche Diagnostics). To generate a DIG-labeled RNA probe that specifically binds to HBV 3.5 kb RNA and its spliced forms, a PCR fragment covering the nt 1998–2447 region was used as templates for in vitro transcription. The signals were detected with CDP-Star reagent (GE Healthcare, Buckinghamshire, United Kingdom). Western blotting was performed as previously described (Li et al., 2016). Briefly, the proteins in cell lysates were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After blocking, membranes were probed with primary antibodies, followed by incubation with peroxidase-conjugated secondary antibody. Antigen-antibody complexes were visualized using ECL Prime Western Blotting Detection Reagent (GE Healthcare).