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. 2007 Aug 8;81(20):11096–11105. doi: 10.1128/JVI.01249-07

FIG. 1.

FIG. 1.

TBC1D20 interacts with the N terminus of NS5A. (A) (Left) A plate from the GAL4-based yeast two-hybrid assay lacking tryptophan and leucine on which yeast cotransformed with the indicated plasmids was plated, selecting for the presence of both the bait (BD; Leu) and prey (AD; Trp) plasmids. LamC, lamin C; T Ag, T antigen. (Right) The same yeast cotransformants, but on a selection plate lacking histidine and adenine, as well as tryptophan and leucine, thus selecting for a reconstituted transcription factor (BD plus AD) mediated by interaction between proteins fused to the respective BD and AD and leading to production of histidine and adenine. This plate demonstrates the specificity of the interaction between NS5A and TBC1D20 and the absence of self-activation with either partner protein alone. See Materials and Methods for additional details. (B) Minimal domains mediating interaction between NS5A and TBC1D20 (circled in red), as determined by interactions of the indicated deletion mutants (the numbers represent the amino acids present in the respective mutants) in yeast two-hybrid assays. +, growth on selective media; green font, noninformative assays due to self-activation of certain mutants. (C) Coimmunoprecipitation of FLAG-tagged TBC1D20 and NS5A from Huh7 cells. Lysates from Huh7 cells cotransfected with FLAG-tagged TBC1D20 and NS5A were immunoprecipitated with anti-FLAG or anti-NS5A antibody, followed by immunoblotting with either anti-NS5A or anti-Flag, respectively. (D) An in vitro-translated AH mutant of NS5A does not interact with TBC1D20. NS5A-FLAG and its AH mutant were in vitro translated in the presence of [35S]methionine. The proteins were then mixed with cold in vitro-translated HA-TBC1D20 and incubated for an additional 30 min at 30°C. HA-TBC1D20 was then immunoprecipitated using an anti-HA antibody. The amounts of labeled wild-type or mutant NS5A were determined by SDS-PAGE analysis, followed by fluorography and exposure to X-ray film. Note that the wild-type, but not AH mutant, NS5A coimmunoprecipitated with TBC1D20 (compare lane 4 to lane 6).