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. 2013 Nov 20;289(1):1–12. doi: 10.1074/jbc.M113.512368

FIGURE 3.

FIGURE 3.

Biotinylation of BAP-tagged Tetherin. A, WB-ra of BAP-tagged Tetherin. Extracts from HEK293T cells were co-transfected with Tetherin-BAP and, where indicated, Vpu in the absence or presence of MG132 (5 μm for 12 h) and developed with anti-SV5. Open and filled arrowheads indicate ER-like glycosylated (ER-glyc) and deglycosylated (de-glyc) Tetherin, respectively, whereas open and filled arrows indicate the corresponding biotinylated (retarded) fractions. B, plasma membrane displayed Tetherin in MG132-treated cells. Membrane-displayed Tetherin was immunoprecipitated with anti-SV5, and the resulting material was digested with Endo-H or PNGase and analyzed in a WB-ra developed with anti-SV5. Cells were co-transfected with Vpu. C, biotinylation of glycosylated and deglycosylated Tetherin. WB-ra shows lysates from cells expressing Tetherin treated with MG132 (5 μm for 12 h). Lysates were treated with Endo-H or PNGase, and the blot was developed with anti-SV5. Open and filled arrowheads and arrows are as in A. D, cyt-BirA biotinylating only retro-translocated molecules. WB-ra shows extracts from cells co-expressing Tetherin-BAP and a scFv BAP-tagged control protein, together with cyt-BirA and treated with MG132 (5 μm) for 12 h. Tetherin was detected with anti-SV5 and the scFv with the anti-roTag. E, cellular fractionation. WB-ra shows total lysates (tot) of cells co-transfected with Tetherin-BAP and cyt-BirA and treated with MG132 for 12 h before lysis and of the pellet (ER) and supernatant (cytosolic, cyt) fractions obtained by ultracentrifugation. Derlin1 and actin were used as ER and cytosolic markers, respectively. F, biotinylation as a non-postlysis event. Cells expressing only Tetherin-BAP (treated with 5 μm MG132 for 12 h) were mixed with cells co-transfected with cyt-BirA and a C-terminal BAP-tagged MHC-Iα (in its cytosolic tail) before mechanical lysis as performed in the cell fractionation experiments. Postnuclear supernatants corresponding to microsomal containing lysates were analyzed in WB-ra. Tetherin was revealed with anti-SV5 and MHC-Iα with anti-roTag. G, WB-ra shows lysates derived from Tetherin-BAP-transfected cells treated with MG132 (10 μm) or chloroquine (50 μm) for 4 h, as indicated. WB of anti-EGFP was used as loading and transfection control.