Fig. 8. Cholesterol-dependent localisation of R-Ras to focal adhesion. Hela cells transfected with pEGFP-R-Ras38V were grown over night in serum-free (K) medium (A,B). Cholesterol replenishment (CD/Chol) was carried out for 30 minutes on EGFPR-Ras38V expressing cells grown overnight in the presence of serum (C,D). Some serum-starved cells were depleted of cholesterol by adding 0.5% ß-methylcyclodextrin (MßCD) for 30 minutes (E,F). (G,H) Cholesterol replenishment of cholesterol depleted cells (CDCD/Chol) for 30 minutes. EGFP-R-Ras38V expressing cells (A,C,E,G) were fixed with paraformaldehyde, permeabilized with 0.1% Triton-X-100 and stained with an antibody against phosphocaveolin (P-Caveolin) (B,D,F,H). Bars, 10 µm.