Identification of β-tubulin as a novel NS1-binding protein.

Abstract

<p>(A) Peptide mass fingerprinting of the 55 kDa protein. The protein was identified as β-tubulin using a program, MASCOT, and the peptides assigned to those of β-tubulin are shown. (B) Confirmation of the 55 kDa protein band in TAP purified protein complexes as β-tubulin. The protein complexes purified from A549 cells transfected with pnTAP-NS1 plasmids and that from pnTAP transfected cells were immunoblotted with the anti-β-tubulin antibody (top panel) or anti-CBP antibody (middle panel). The total cell lysate was also immunoblotted with the anti-CBP antibody (bottom panel). (C) Co-immunoprecipitation analysis of β-tubulin and NS1. The precipitates obtained were immunoblotted with the anti-β-tubulin antibody (top panel) or anti-CBP antibody (middle panel). The total cell lysate was immunoblotted with the anti-CBP antibody (bottom panel). (D) An illustration of the various NS1 truncations used to map the β-tubulin-binding domain in the NS1 protein. NS1 Full (NS1 full-length); NS1 N (NS1 N- terminal domain, that is the RNA binding domain); and NS1 C (NS C-terminal domain, that is the effector domain). (E–F) The N-terminal domain of NS1 interacts with β-tubulin. Left panel: the purified GST-fused NS1 proteins stained by coomassie brilliant blue. Right panel: the purified GST-fused NS1 proteins complexes obtained were detected by immunoblotting with anti-GST antibodies (top panel) or anti-β-tubulin antibodies (bottom panel).</p