Vpu impairs recycling of CD1d to the cell surface. (A) 293T cells stably expressing wt CD1d or mutants CD1dY331A or CD1dΔtail, respectively, were transiently transfected with Vpu-EGFP. CD1d down-regulation was calculated 48 hours after transfection. Data are representative of 5 independent experiments. Error bars represent SD; ***P < .001. (B) The rate of CD1d internalization in 293T-CD1d cells expressing Vpu-EGFP or EGFP alone was measured 48 hours after transfection with the use of a flow cytometry–based assay as described in “Recycling assay.” Data are representative of 6 independent experiments. Error bars represent SD. (C) For flow cytometric analysis of CD1d recycling, 293T-CD1d cells transfected with Vpu-EGFP were pretreated with purified anti-CD1d mAb to block surface CD1d. After incubation at 37°C for different times, the surface arrival of unblocked internal CD1d was detected by staining with a phycoerythrin-conjugated CD1d antibody. Recycling of CD1d was measured as an increase in cell surface CD1d MFI and analyzed separately for Vpu-EGFP⁺ (closed circles) and Vpu-EGFP⁻ (open circles) cells in the same culture. Data from 1 representative of 4 independent experiments are shown. All experiments were performed in duplicates. (D) Quantification of CD1d recycling rates in Vpu-EGFP⁺ and Vpu-EGFP⁻ cells. The rate of recycling is expressed as an increase in CD1d MFI per minute and corresponds to the slope of the regression line in panel C. Data from 4 independent experiments performed in duplicates are shown. **P < .01.