Colocalization and interaction of tetherin/BST-2 with P2Y₁₂R. Tetherin/BST-2 colocalized with the P2Y₁₂R in (A-B,D) mouse platelets and (B-C) HEK293 cells. (A) Representative confocal images of permeabilized mouse platelets spread on fibrinogen and treated with rat anti-mouse tetherin/BST-2 and rabbit anti-P2Y₁₂, followed by anti-rat Alexa 488 (green)– and anti-rabbit Alexa 546 (red)–conjugated secondary antibodies. Bar represents 10 µm. (B) Pixel intensity histograms of gray scale images (A; bottom) corresponding to output from green and red channels (A; top). (C) HEK293 cells were transiently transfected with tetherin/BST-2-GFP alone or with either mCherry or mCherry-HA-P2Y₁₂R. Fixed cell samples were prepared for subsequent confocal microscopy. Representative confocal images of HEK293 cells coexpressing BST-2-GFP (green) and mCherry (red; top), GFP and mCherry-P2Y₁₂-HA (middle), or BST-2-GFP and mCherry-P2Y12-HA (bottom). Bar represents 1 μm. Images from panels A and C underwent subsequent quantitative analysis (C; n = 16 for platelet and n = 22 for HEK293 cells, across 3 independent experiments). (D) Pearson’s correlation coefficients (PCCs) in either platelets or in the whole cell vs the membrane or cytoplasm of the cell. The PCCs were significantly higher in membranes vs either the whole cell or cytoplasm. *P < .05; Student t test. (E-F) HEK293 cells cotransfected with mCherry-HA-P2Y₁₂ and either tetherin/BST-GFP or GFP control were treated with ADP (20 μM; 0-60 minutes). The cells were lysed, and the receptors were immunoprecipitated with monoclonal mouse anti-HA-agarose. (E) The samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted for associated anti-GFP (top) and reprobed with an anti-HA antibody to show the total receptor immunoprecipitated (second gel). Inputs were immunoblotted for GFP (third gel) and anti-α-tubulin to show equal protein loading. Gels are representative of 3 independent experiments. (F) Quantification of tetherin/BST-2 coimmunoprecipitated with P2Y₁₂R. Data are normalized relative to association of tetherin/BST-2 with P2Y₁₂R at rest vs agonist stimulated and expressed as means ± standard error of the mean (n = 3; *P < .05, Mann-Whitney U test). (G) The P2Y₁₂R was immunoprecipitated from control or ADP-treated, washed platelets (10 µM; 30 minutes) with a receptor-specific rabbit antibody, as outlined in “Methods.” Immunocomplexes were resolved by 10% SDS-PAGE and immunoblotted with anti-BST-2 antibody (top gel). Inputs were immunoblotted for P2Y₁₂R (second gel), BST-2 (third gel), and anti-α-tubulin (bottom gel) to show equal protein loading.