Figure 8
From: Human Polycomb groupEED protein negatively affects HIV-1 assembly and release
Cellular distribution of EED3/4 upon NEF expression. (a), Experimental protocol. Cells were cotransfected with pNL4-3Luc(R-E-) and pTracer-EED (or pTracer-Emp), with or without coexpression of various Nef proteins, WTNef or NefG2A and NefΔ57 mutants, or fusion constructs LAT-Nef or LATAA-Nef, as indicated on top of each panel. Cells were fractionated into cytosolic supernatant (C), membrane fraction (M) and insoluble pellet (P), as shown in panels (b),(c),(e) and (f). Fractions were probed for (b) Gag, (c, d, f) EED, (e) Nef, and (d) CD55. (d), Isolation of lipid rafts by ultracentrifugation of flotation. Gradient fractions were analyzed by SDS-PAGE and immunoblotting, using anti-CD55 antibodies (detected by phosphatase-labeled complementary antibody) and anti-EED antibodies (detected by peroxidase-labeled complementary antibody). (m), Protein markers, with molecular masses indicated in kDa. Bands of exogenous EED3 and EED4 isoforms are indicated by black dots. Note that EED did not cosediment with lipid rafts, identified by the CD55 marker. Coexpression of EED and WTNef, NefG2A or LATAA-Nef, but not NefΔ57 or LAT-Nef, resulted in the relocation of EED and Nef proteins in a cellular compartment recovered as pelletable fraction (P).