FIG 4.

T242 is exclusively phosphorylated in the hyperphosphorylated form of NS5A. (A) Different concentrations of peptide either phosphorylated at position T242 or nonphosphorylated were titrated on PVDF membrane and incubated with purified, polyclonal anti-pT242 antibodies. (B) Huh7-Lunet T7 cells were either transfected with a pTM vector encoding JFH-1 NS3-3′ or electroporated with in vitro transcripts encoding a bicistronic reporter replicon (SGR JFH-1), a full-length reporter virus genome (JcR2a), or an unmodified full-length virus genome (JC1). The cells were lysed 24 h posttransfection or 72 h postelectroporation, respectively, and analyzed by 7.5% SDS-PAGE/Western blotting using anti-NS5A (monoclonal 9E10)-, anti-NS5A-pT242-, anti-NS3-, and anti-calnexin-specific antibodies. Shown is one representative experiment (n = 2). Note the discrepancy between the 58-kDa molecular weight (MW) marker band (left) and the band referred to as p58/pT242 (right), which is due to a higher apparent MW of the NS5A phosphoisoforms in the case of JFH-1.