Staufen1 co-fractionates with Gag in lipid rafts. (A) A detergent-free method for the isolation and fractionation of lipid rafts. HeLa cells were mock transfected (B) or co-transfected with pCMV-Rev and Rev-dependent Gag-VN and Gag-VC (C) or Rev-dependent GagΔNC/p6 (D). At 24 hr post-transfection, cells were harvested and fractionated on Optiprep gradients: lipid rafts fractionated in fractions #2-6 and non-membrane associated proteins in the bottom fractions. Western blotting identified Caveolin-1 (Cav-1), Staufen1 (two isoforms: St-55, St-63), IMP1, TSC2, Gag-VC (in C) and p24 (to detect GagΔNC/p6 in (D)) in each fraction. (E) The relative quantities of Staufen1 in each fraction were measured using ImageJ software (NIH) (the sum of all fractions per condition = 1.0).