Deletion analysis of DEN NS4B. (A) Schematic representation of various deletions of DEN-2 NS4B created by PCR. The N-terminal 2K segment was deleted (Δ2K-NS4B), left intact (NS4B), or replaced by the murine MHC-I signal peptide KB (KB-NS4B). In addition, deletions of the C-terminal region were also created by PCR [NS4B(1-232), NS4B(1-155), NS4B(1-125), NS4B(1-77), and NS4B(1-47)] as indicated. (B) Immunoblot analysis of HA-tagged NS4B derivatives. All fragments represented in panel A were cloned in pCAGGS-HA to tag the C-terminal end of each protein. Expression levels of each protein were examined by running transfected cell extracts in a 4 to 20% gradient polyacrylamide. Proteins were transferred to a nitrocellulose membrane and detected with anti-HA primary antibody and horseradish peroxidase-labeled secondary antibody. Molecular mass (kDa) markers are at left.