Figure 7.

Cholesterol depletion by methyl-β-cyclodextrin (CyDx) and its effect on cell surface density of CKRs. (A) Cholesterol staining of CyDx-treated HeLa cells. After treatment, cells were fixed and stained for cholesterol with fillipin (10 μg/ml) for 10 min at RT and visualized by UV laser microscopy. (B) FACS histograms of CKR expression in HeLa cell transfectants treated with CyDx (5 mM) or fillipin (10 μg/ml) or left untreated. CD4 was cotransfected in each case to monitor expression. Transfectants were stained with CD4-APC and PE-conjugated mAbs for the respective receptors, except for the KRFX mutant. Because transport of this mutant to the cell surface was quite poor, cells were stained with unconjugated 1^o mAbs followed by PE-conjugated 2^o antibodies. Cells were gated for CD4 staining because it was not altered by cyclodextrin or fillipin treatment. Histogram profiles of CKR densities in CD4-gated populations representing two experiments are shown. Shaded histograms represent staining with an isotype control.