Fig. 2.

The use of SILAC to determine the origin of proteins in DRMs. A: Cells were grown in three SILAC media separately containing normal isotopic abundance Lys and Arg (0/0), ²H₄-Lys and ¹³C₆-Arg (4/6), or ¹³C₆¹⁵N₂-Lys and ¹³C₆¹⁵N₄-Arg (8/10). Mitochondria from both 0/0 and 4/6 cell populations were isolated (see Materials and Methods), and then 0/0 mitochondria were treated with the cholesterol-disrupting drug MβCD. Afterwards, 0/0 and 4/6 mitochondria preparations were solubilized in Triton X-100. At the same time, whole 8/10 cells were also solubilized with Triton X-100. Equal amounts of protein from all three extracts were mixed prior to isolation of DRMs by floatation on a sucrose density gradient, and then the proteins were subsequently analyzed by LC-MS/MS. B: True mitochondrial raft proteins should have high 4/6:0/0 ratios indicating their sensitivity to cholesterol disruption, while high 4/6:8/10 ratios would indicate that proteins are coming from mitochondria.