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. 2010 Nov 10;85(2):932–945. doi: 10.1128/JVI.01668-10

FIG. 2.

FIG. 2.

Interaction of tetherin and Vpu via TM domains. (A) The KGN-Vpu-expressing cells were transfected with plasmid encoding KGC-tagged hu-tetherin or deletion mutants of CT or GPI (delCT and delGPI). The expression of KGC-tetherin fusion protein was detected using an anti-KGC antibody (bottom). Relative MFI is defined as the MFI of pKGC-tetherin-transfected or its mutant-transfected cells minus the MFI of untransfected cells, and results represent the means from three independent experiments plus standard deviations. The amount of protein applied for the delGPI Western blotting was 10 times less than that for WT hu-tetherin to avoid the overexposure of the protein on polyvinylidene difluoride membrane. Statistical significance (Student's t test) in the MFI of human tetherin and mutants are represented as follows: **, P < 0.01; *, P < 0.05. (B) The KGN-Vpu-expressing HEK 293 cells were transfected with pKGC-mo-tetherin (upper) or pKGC-hu-tetherin (lower). Cells were double stained with anti-Vpu antibody and anti-KGC antibody and imaged by confocal microscopy. (C) Interaction of tetherin TM with Vpu. The KGN-Vpu-expressing cells were transfected with KGC-hu-tetherin, KGC-mo-tetherin, or chimera mutant DNA containing reciprocal exchanges of the TM, ECL, CC, or GPI between the hu- and mo-tetherin proteins and analyzed by flow cytometry. Statistical significance (Student's t test) in the MFI of hu-tetherin and chimeras are represented as follows: *, P < 0.01; standard deviation for mo(huCC) was 0.0061. Relative MFI is defined as the MFI of pKGC-tetherin-transfected or its mutant-transfected cells minus the MFI of untransfected cells. (D) Interaction of the Vpu TM with tetherin. KGC-tetherin-expressing cells were transfected with KGN-Vpu or its mutant DNA and analyzed. Statistical significances (Student's t test) in the BiFC signals are given. Relative MFI values are defined as the MFI of pKGN-Vpu or its mutant-transfected cells minus the MFI of untransfected cells. The results represent the means from three independent experiments (A, C, and D). Expression of the KGC or KGN fusion or Vpu protein was detected by immunoblot analysis with individual antibodies (A, C, and D).