Tetherin/BST-2 interacts directly with the P2Y₁₂R. HEK293 cells were transiently transfected with tetherin/BST-2-GFP (FRET donor) alone or with either mCherry or mCherry-HA-P2Y₁₂R (FRET acceptor). Cells coexpressing tetherin/BST-2 and P2Y₁₂R were stimulated with ADP (20 μM; 0-60 minutes) or with ADP (20 μM; 60 minutes), followed by the addition of apyrase (1 U/mL) for 5 minutes. Fixed cell samples were prepared for subsequent microscopy. (A) Representative fluorescence intensity (gray scale images, top) and corresponding intensity-weighted (pseudocolor, bottom) images of cell membranes at selected time points of measurement. (B) Fluorescence life measurements of tetherin/BST-2-GFP from cell membrane regions showing association of P2Y₁₂R with tetherin/BST-2. Coexpression of tetherin/BST-2-GFP (donor) with mCherry-HA-P2Y₁₂R (acceptor) significantly reduced donor vs mCherry life (*P < .05; Student t test) indicative of protein proximity. Addition of ADP (5, 30, and 60 minutes) significantly increased donor life vs the non–agonist-treated control (§P < .05; Student t test) or after apyrase removal of ADP (€; P < .05; Student t test). (C-E) Agonist-induced changes in P2Y₁₂R and tetherin/BST-2 intracellular expression were assessed, and the data were normalized and expressed as a percentage of whole-cell fluorophore intensity. (C) Cell images captured before FLIM were segmented in CellProfiler by using a semiautomatic approach. Identified objects were subsequently reduced in size by 10% to produce complementary masks to define the cell edge and central regions. ADP treatment had no effect on tetherin/BST-2 intracellular expression (D), but promoted a significant increase in intracellular accumulation of the P2Y₁₂R vs non–agonist-treated control (*P < .05; Student t test) or after apyrase removal of ADP (P < .05; Student t test; E). (B,D-E) Data points represent a single cell (n > 12 per transfection condition) pooled across 3 independent experiments with line and error bars representing means ± standard error of the mean .