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Figure 5 | Retrovirology

Figure 5

From: The formation of cysteine-linked dimers of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for sensitivity to Vpu

Figure 5

BST-2 forms cysteine-linked dimers. (A) HeLa cells were lysed in reducing (+β-ME) or non-reducing (-β-ME) sample buffer as described in Methods. Lysates were separated by SDS-PAGE and subjected to immunoblot analysis using a BST-2-specific polyclonal antibody. (B) Schematic representation of mutants analyzed in this experiment. In all cases, cysteine residues were mutated to alanine by site-directed mutagenesis. Remaining cysteine residues are shown for each mutant. (C) 293T cells were transfected with wt BST-2 (lanes 1 & 6), the triple cysteine mutant C3A (lanes 2 & 7), or individual cysteine mutants (lanes 3-5 & 8-10). Cells were harvested 24 h post transfection, washed in PBS, and split into two equal samples. One set of samples was mixed with an equal volume of reducing sample buffer (lanes 1-5); the second set was mixed with sample buffer lacking β-ME (lanes 6-10). Cell lysates were separated by SDS-PAGE and subjected to immunoblotting using a BST-2 specific antibody. (D) 293T cells were transfected with wt BST-2 (lanes 1 & 6), the triple cysteine mutant C3A (lanes 5 & 10), or double-cysteine mutants (lanes 2-4 & 7-9). Samples were analyzed under reducing and non-reducing conditions as in panel C. (HM) marks the position of the high-mannose forms of BST-2 in the gels.

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