FIG. 5.

PLD activity is required for Ras and ERK activation in response to cholesterol depletion. BHK cells were treated with lovastatin for 48 h in the presence of 10% LPDS. Control cells were cultured in 10% DCS. For the final 4 h of incubation, n-butanol (BtOH) or t-BtOH was added to the growth medium to give a final concentration of 1% (vol/vol). Ras-GTP levels were measured using an RBD pulldown assay (a) and ppERK levels measured by quantitative immunoblotting (b). A representative blot for each experiment is shown, with total Ras and ERK2 used as loading controls. Growth in n-butanol inhibits phosphatidic acid production by PLD, whereas growth in t-butanol does not (Materials and Methods). The graphs show mean levels ± SEM for 3 independent experiments. Differences between lovastatin-treated and control cells grown in n-BtOH or t-BtOH were assessed using one-way ANOVA tests. Significant differences are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001).