CD1d is down-regulated from the cell surface by HIV-1 Vpu. (A-B) 293T cells stably expressing CD1d were transiently transfected with EGFP or Vpu-EGFP and examined for CD1d cell surface expression 48 hours after transfection. Dashed black lines indicate EGFP⁺ cells; solid black lines, EGFP⁻ cells in the same culture; gray solid lines, isotype control stainings. CD1d down-regulation was calculated as described in “Flow cytometry and surface protein down-regulation.” Data are representative of 8 independent experiments. Error bars represent SD; ***P < .001. (C) Surface immunofluorescence microscopy of 293T cells 48 hours after cotransfection with CD1d and Vpu-EGFP. Arrowheads indicate representative EGFP⁺ cells; asterisks, representative EGFP⁻ cells. Original magnification of the images, ×630; scale bar, 15 μm. (D) Forty-eight hours after transfection, 293T-CD1d cells transfected with Vpu-EGFP were stained for surface expression of CD59 or CD71. Dashed black lines indicate EGFP⁺ cells; solid black lines, EGFP⁻ cells; gray solid lines, isotype controls. Representative data from 1 of 3 experiments are shown.