Complex formation of CD1d and Vpu. (A-B) Confocal microscopy analysis of 293T cells transfected with CD1d alone (A left) or cotransfected with CD1d and Vpu-EGFP (A right, B). Cells were permeabilized and stained with anti-CD1d antibody. Original magnification of all images, ×630; insets at higher magnification. Scale bars, 15 μm. (C) 293T cells were cotransfected with CD1d and Vpu-EGFP or EGFP alone and lysed in buffer containing 1% Igepal. After immunoprecipitation with anti-GFP microbeads, proteins were separated on a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and transferred onto a nitrocellulose membrane. The membrane was first probed with anti-CD1d antibody (clone NOR3.2) and subsequently stripped and reprobed with anti-GFP antibody.