Skip to main content
. 2016 Apr 22;6:24934. doi: 10.1038/srep24934

Figure 4. Domains of SGTA, Vpu and tetherin required for the SGTA-induced accumulation of non-glycosylated tetherin.

Figure 4

(A) Truncated SGTAs that were tested to identify the domain of SGTA required for accumulation of non-glycosylated tetherin include the tetratricopeptide containing domain (TPR); the C-terminal domain (Ct); a mutant lacking the N-terminus (delN); the N-terminal domain (Nt); and the N-terminal plus TPR domains (N200). (B) 293T cells were transfected with the vector expressing HA-tagged tetherin with or without Vpu and FLAG-tagged SGTA expression vectors. One day posttransfection, cells were lysed and subjected to western blot analysis with anti-HA to detect HA-tagged tetherin or anti-FLAG to detect FLAG-tagged SGTA or anti-Vpu antisera. Mobility of molecular mass standards is shown on the right of anti-HA and anti-FLAG blots. The location of the non-glycosylated, 23-kDa tetherin species is indicated by the arrow. (C) 293T cells were transfected with vectors expressing FLAG-tagged SGTA, HA-tagged tetherin, and WT or mutant Vpu (RKR-AAA, R30A, K31A, R34A, and SS-AA). One day posttransfection, cells were lysed and subjected to western blot analysis with anti-HA antibodies to detect HA-tagged tetherin or anti-FLAG antibodies to detect FLAG-tagged SGTA or anti-Vpu antisera. Molecular mass markers are shown on the right of the anti-HA blot. The location of the non-glycosylated, 23-kDa tetherin species is indicated by the arrow. (D) 293T cells were transfected with vectors expressing HA-tagged tetherin and Vpu with or without FLAG-tagged SGTA expression vector. Tetherin variants tested to identify the regions of tetherin required for SGTA-mediated stabilization of non-glycosylated tetherin include a cytoplasmic tail deletion mutant (delCT); a GPI-anchor deletion mutant (delGPI); Agm tetherin (Agm); and a human tetherin containing the Agm transmembrane domain (Hu-Agm). One day posttransfection, cells were lysed and immunoblotted with anti-HA antibodies to detect HA-tagged tetherin or anti-FLAG antibodies to detect FLAG-tagged SGTA or anti-Vpu antisera. Mobility of molecular mass standards is shown on the right of anti-HA blot. The location of the non-glycosylated, 23-kDa tetherin species is indicated by the arrow.