FIGURE 1:

Budding of APOB lipoprotein cargoes in a cell-free reaction. (A) Scheme of cell-free vesicle budding reaction. In brief, McArdle RH7777 cells were incubated with oleic acid. Treated cells were used to prepare donor membranes which were then incubated at 37°C with GTP and purified recombinant human COPII proteins. McArdle membranes were washed with high salt, the high-salt wash (HSW) was dialyzed and added to a vesicle budding reaction where indicated, heated (“H”) or treated with proteinase K, then heated (“K”) as indicated. Vesicles in 18,000 × g supernatant fractions from budding reactions were isolated by density gradient flotation. (B) Fractions from the top of an OptiPrep gradient were analyzed by immunoblot. APOB serves as a marker for large VLDL cargoes and ERGIC53 serves as a marker for small traditional COPII cargoes. Ribophorin serves as a marker for ER contamination.