Simvastatin treatment reduces the cholesterol content of lipid rafts of LNCaP cells and inhibits Akt phosphorylation in rafts. (A) Immunoblot results obtained following fractionation of Triton X-100–insoluble material by sucrose gradient ultracentrifugation. This panel demonstrates how lipid raft fractions used for the cholesterol determinations shown in B were obtained. Flotation fractions demonstrating enrichment in the raft markers G_iα2 and flotillin-2 (i.e., fraction 6 in this example) were designated as raft fractions. (B) Cells were incubated in serum-free medium in the absence (control) or presence of 10 μM simvastatin overnight at 37°C. After the drug treatment, 1 group was incubated with cholesterol complexes (sim + chol) at 37°C for 1 hour. The cholesterol/protein ratio was determined in lipid raft fractions prepared as shown in A and under the conditions described in the text. Values shown are means ± SD of triplicate determinations (*P < 0.01). (C) Cells were incubated in the presence of 20 μM simvastatin or vehicle in serum-free medium at 37°C for the indicated times. C+M and raft fractions were isolated by successive detergent extraction, resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies. (D) Cells were treated with 20 μM simvastatin with or without cholesterol complexes for 4 hours, followed by raft extraction and analysis as in C.