Multiple roles of PP2A binding motif in hepatitis B virus core linker and PP2A in regulating core phosphorylation state and viral replication
Fig 10
Effects of PP2A inhibitors on HBc dephosphorylation in vitro.
The WT and 3E mutant HBc proteins were translated in RRL. The translated HBc, without any further treatment (untreated) or treated with NEBuffer 3 plus CIAP overnight at 37°C (CIP), with NEBuffer 3 plus a mixture of non-specific phosphatase inhibitors over night at 37°C (PPI), NEBuffer 3 alone overnight at 37°C (mock 1), with NEBuffer 3 plus 0.01% DMSO overnight at 37°C (CIAP) (mock 2), with NEBuffer 3 plus increasing concentrations of Fostriecin (10 nM, 20 nM, 40 nM, 80 nM) or Okadaic acid (1 nM, 10 nM, 100 nM), before further analysis. A. The 35S-labeled HBc proteins (WT and 3E) were resolved by SDS-PAGE and detected by autoradiography or western blot analysis using the HBc CTD dephosphorylation specific mAb 25–7. B. The HBc 3E reactions were resolved by NAGE. The 35S-labeled HBc proteins were detected by autoradiography (total HBc) or western blot analysis using the HBc specific polyclonal antibody (Dako). C. The HBc 3E reactions were resolved by Phos-tag SDS-PAGE, with the no-template translation reaction as a negative control, and 35S-labeled HBc proteins were detected by autoradiography. a & b: hyperphosphorylated 3E; c-e: hypophosphorylated 3E; f: non-phosphorylated 3E.