FIGURE 1:

Schematic of fluorescence method to study sterol transport between plasma membrane and ERC. U2OS-SRA cells are loaded with DHE complexed to MCD for 1 min. DHE is allowed to equilibrate among cellular membranes for 120 min. At steady state, DHE is enriched in the ERC and plasma membrane (yellow). The ERC is identified using fluorescently labeled Tf (red outline). (A) To monitor transport to the ERC, the ERC is photobleached, and the FRAP of DHE is recorded. (B) To monitor transport from the ERC, cellular medium is exchanged with medium containing red blood cells (RBCs) and HPCD–cholesterol complex. As fluorescent DHE is exchanged at the plasma membrane for nonfluorescent cholesterol, the fluorescence signal of the ERC decreases over time.