Fig. 4.
The preferential interactions of activated N-Ras with rafts and its shift to exchange in response to HA-GPI cross-linking are retained at 37°C. COS-7 cells expressing GFP–N-Ras (wt or G13V) alone or with HA-GPI were subjected (or not) to cholesterol depletion, followed by HA-GPI cross-linking (or Fab′ labeling) and FRAP beam size analysis at 37°C as described in the Fig. 2 legend. Bars show means ± SEM (n = 30 to 60). (A and B) Effects of cholesterol depletion. Comparison between τ values (A) measured with the same beam size shows that cholesterol depletion significantly reduces the τ of GFP–N-Ras(G13V) (***, P < 10−8; Student's t test), with a milder effect on GFP–N-Ras(wt) (**, P < 10−5). (C and D) HA-GPI cross-linking induces a cholesterol-dependent shift of N-Ras(G13V) to exchange. The significant effects of HA-GPI cross-linking on the τ(40×) of N-Ras(G13V) (***, P < 10−8; Student's t test) (C) and on its τ ratio (***, P < 10−12; bootstrap analysis) (B) were abolished in cholesterol-depleted cells.