FIG. 4.

Cell type-dependent sensitivity of VP4 association with DRM to cholesterol extraction does not depend on caveolin expression. (A) Caco-2 cells stably transfected with caveolin-1 (Cav-1-Caco-2) were cultured for 10 days after reaching confluence. Transfected caveolin-1 distribution along a sucrose gradient after cell lysis in TX-100 at 4°C is shown. Note that wild-type Caco-2 cells do not express any detectable Cav-1 (not shown). (B) Cav-1-expressing and wild-type Caco-2 cells were radiolabeled with cholesterol and infected with rotavirus (10 PFU/cell). They were not treated or treated with 10 mM CD before cell lysis, gradient separation, and DRM collection. Equal amounts (in volume) of floating materials (DRM) and soluble fractions (Sol) were collected and analyzed for VP4 and flotillin (Flot) content by SDS-PAGE. (C) [³H]cholesterol, VP4, and flotillin were quantified and expressed as described in the legend to Fig. 2. Data are the means ± standard deviations for two different experiments. Gray bars, untreated cells; black bars, CD-treated cells.