Figure 3.

Expression of glycosylated E2 inhibits HIV-1 Gag membrane targeting. The Gag-Pol expression vector was cotransfected with empty vector or with E2 or immunoglobulin G (IgG)–E2 expression vector. Two days after transfection, 293T cells were sonicated. Nuclear fractions and unlysed cells were removed by centrifugation. The clear postnuclear supernatants were mixed with 85.5% sucrose to adjust the sample to 2 mL with 73% sucrose. The sample was then placed at the bottom of a centrifuge tube and layered with 7 mL of 65% sucrose in Tris–ethylenediaminetetraacetic acid (TE) and 3.25 mL of 10% sucrose in TE. The sucrose step gradient was centrifuged at 35 000 rpm for 18 hours at 4°C in a Beckman SW41 rotor. Eight fractions were collected from each sample after ultracentrifugation. The fraction samples and total cell lysates of Gag-Pol cotransfected vector (Figure 3A), E2 (Figure 3B), or IgG-E2 (Figure 3C) were analyzed by Western blot.