BRET assay to monitor BST-2/Vpu interaction. (a) Western blot analysis of cellular lysates following the cotransfection of BST-2 and Vpu or Vm plasmids into HEK 293 T cells. (b) Schematic representation of the plasmid DNA constructs. R-B and E-B were constructed with either Rluc or EYFP fused to the N terminus of BST-2; B-R and B-E were constructed with either Rluc or EYFP fused to the C terminus of BST-2; The N-terminus of Vm was fused with Rluc and EYFP to generate R-Vm and E-Vm; The C-terminus of Vpu was fused with Rluc and EYFP to engineer Vm-R and Vm-E. (c) Expression of the fusion proteins. Western blot analysis of the cellular lysates following the transfection of the expressing plasmid into HEK 293 T cells using anti-BST-2 and Vpu antibodies. (d) The BRET signals of BST-2 and Vpu interaction. HEK293T cells were transfected with different combinations of donor and acceptor DNA constructs. The BRET ratio was measured using a Pekin Elmer reader followed by the addition of substrate Coelenterazine at the final concentration of 5 μM. (e) The schematic diagram of the energy transfer between R-B and V-E. (f) Subcellular localization of R-B and Vm-E. Immunofluorescence staining was performed to detect the localization of BST-2(red), Vpu (green), R-B(red) and Vm-E(yellow) expressed in HEK 293 T cells. The nuclei were stained with DAPI (blue). Representative images are shown. (g) Function analysis of the tagged BST-2 and Vpu proteins. Western blot analysis of the cellular lysates and the corresponding viral lysates following cotransfection of the HIV-1 Gag-Pol construct (containing gag and pol genes) and other expressing plasmids into HEK293T cells using anti-BST-2, anti-Vpu, anti-actin and anti-p24 antibodies.