FIG. 3.

Expression of hIL-2::GPI on 293 producer cells and VLP. (A) GPI anchor attachment targets hIL-2 to lipid rafts. 293 cells transfected with hIL-2::GPI, hIL-2:TM(CD80), or hIL-2::TM(CD99) were lysed at 4°C with 1% Triton X-100 and fractionated on 5 to 40% sucrose into nine fractions (top to bottom). Equal amounts of each fraction were resolved by SDS-PAGE, blotted, and probed (immunoblotting) with the hIL-2-specific mAb IL-2.1E7. Bound antibody was visualized by goat anti-mouse Ig conjugated to HRP. (B) Preferential targeting of hIL-2::GPI to VLP. 293 cells were transfected with the indicated cDNAs or control vector and cell lysates (cells), corresponding VLP preparations, as well as 30 ng of soluble (sol.) recombinant hIL-2 and were analyzed by SDS-PAGE under reducing conditions followed by immunoblotting (IB) with the hIL-2-specific mAb IL-2.1E7. One out of several typical experiments is shown. (C) As a control for proper vesicle induction and loading in B, viral core protein (p30 Gag) expression was determined in cell lysates (cells) and VLP. (D) VLP isolated from the cell culture supernatant of producer cells cotransfected with MoMLV gag-pol and hIL-2::GPI (left micrograph) or MoMLV gag-pol and control vector (right micrograph) were put onto Formvar-carbon-coated electron microscopy grids, fixed, and immunogold labeled by incubation with the hIL-2-specific mAb IL-2.1E7 followed by rabbit-anti-mouse Ig and protein A coupled to 10-nm gold particles and contrasted as described in Materials and Methods.