Figure 1
From: Amphotropic murine leukaemia virus envelope protein is associated with cholesterol-rich microdomains
A-MLV envelope protein associates with detergent resistant microdomains (DRMs). A) 293T cells producing A-MLV were treated with TX-100 at 4°C and loaded on a discontinuous sucrose gradient. Western blot analyses were performed. Fraction 1 corresponds to the top and fraction 6 corresponds to the bottom of the tube. Fractions 1 to 4 contain the DRMs, fractions 5 to 6 the non-DRM membrane fractions. A-MLV Env is found predominantly in the DRM fractions 2, 3 and 4. EGFP, which is localized in the cytoplasm, remains in the soluble fractions 5 and 6. B) NIH3T3 cells releasing A-MLV were treated with TX-100 at 4°C and loaded on a discontinuous sucrose gradient. Dot blot analyses were performed. Fraction 1 corresponds to the top and fraction 6 corresponds to the bottom of the tube, respectively. B is the background of the dot blot. Fractions were processed in parallel for immunological detection of cav-1 and A-MLV Env. C) Quantification of the dot blot shown in B) using image analysing software. The amounts shown are determined as percentages of the total of all dots; DRM (fractions 1 to 3), non-DRM (fractions 4 to 6). D) Detergent soluble supernatant (non-DRM) and insoluble pellet (DRM) of A-MLV producing NIH3T3 cells treated with TX-100 at 4°C or 37°C were investigated for the amount of envelope protein using dot blot analysis. The results of two independent experiments are shown. The amounts shown are determined as percentages of the total of all dots.