TBC1D20 overexpression interferes with the association of HIV Env with DRMs. (A) Timeline of experimental procedure (CHK, cyclohexamide). (B) Equilibrium flotation centrifugation assay to determine gp41 distribution in soluble and detergent-resistant cellular fractions. Cells (~107 cells/10 cm) overexpressing the indicated GAPs (fused to GFP) or GFP were fractionated and analyzed by Western blotting, using anti- gp41 and caveolin antibodies. Top and Bottom designate the top and bottom gradient fractions, respectively. The ratio between gp41 levels in the soluble fractions (fractions 9-12) and in the detergent-resistant fractions (fractions 4-7) was determined by densitometry (C), and is represented as the mean ± SEM (n = 3).