Figure 3

In vitro and in vivo interaction between hnRNPC and S-HDAg. (A) Overlay of bacterially expressed His₆/hnRNPC with recombinant S-HDAg. E. coli protein extracts were prepared after induction of recombinant protein expression with IPTG. Increasing amounts of His₆/hnRNPC-containing extracts (lanes 2 to 6), were separated by SDS/PAGE, blotted onto nitrocellulose membranes and overlaid with the same amount of purified recombinant S-HDAg. Detection of S-HDAg was performed with a specific rabbit polyclonal antibody. E. coli protein extracts lacking His₆/hnRNPC (lane 1) were used as a negative control (upper panel). The presence of His₆/hnRNPC in the extracts was confirmed using a mouse anti-His-tag monoclonal antibody (middle panel). In the lower panel, the assay was performed in the absence of purified S-HDAg showing that the anti-HDAg polyclonal antibody does not recognize hnRNPC. (B) Co-immunoprecipitation of hnRNPC with S-HDAg. Huh7-D12 cell lysates were immunoprecipitated with an anti-HDAg antibody bound to protein G beads. The immunoprecipitates were separated on 12% SDS/PAGE gels and immunoblotted with an anti-hnRNPC antibody. The negative controls were performed by incubation of Huh7-D12 lysates with beads protein G beads and by immunoprecipitating Huh7 lysates with an anti-HDAg antibody.