Figure 6
From: The Vpu-interacting Protein SGTA Regulates Expression of a Non-glycosylated Tetherin Species
The non-glycosylated tetherin species partially localizes to the cytosol.
(A,B) 293T cells were transfected with the vector expressing HA-tagged tetherin with or without Vpu and FLAG-tagged SGTA (WT and individual domains) expression vectors. (A) twenty-four h post-transfection, cells were fixed and co-stained with either anti-HA, anti-Vpu and anti-FLAG, or anti-HA and anti-calnexin antibodies and images were acquired with a Delta-Vision deconvolution microscope. (B) cells were fixed after 24 h post-transfection and co-stained with anti-HA, anti-Vpu and anti-FLAG antibodies. Scale bars in (A,B) are identical and represents 15 μm. (C) 293T cells were transfected with vectors expressing Vpu alone or with HA-tagged tetherin in the presence and absence of SGTA. Twenty-four h post-transfection cells were sonicated and membrane and cytosolic fractions were separated by ultracentrifugation and immunoblotted with anti-HA antibodies to detect HA-tagged tetherin or anti-FLAG antibodies to detect FLAG-tagged SGTA or antibodies specific for Vpu or transferrin receptor. Molecular mass markers are shown on the right of the anti-HA blot. (D) 293T cells were transfected with vectors expressing WT tetherin or the non-glycosylated (NN-AA) tetherin mutant with or without Vpu and FLAG-tagged SGTA expression vectors. The cytosol and membranes were fractioned as in (C) and immunoblotted with anti-HA antibodies to detect HA-tagged tetherin. The location of the non-glycosylated, 23-kDa tetherin species is indicated by the arrow.
