HIV-1 Gag and Env exhibit altered association with lipid rafts in Ubc9 knockdown cells.
(a) Lipid raft isolation. Representative immunoblot of sucrose fractions for lipid raft markers Flotillin-1 and non-raft marker transferrin receptor 1 (TfR). Fractions containing lipid raft markers (LR), detergent soluble membranes (DSM; raft excluded), and detergent resistant membranes (DRM; non DSM). (b) Average density of all sucrose fractions prior to being adjusted to 1X lysis buffer. 293T cells were transfected with pNL4-3 alone (c), or in combination with either Ctr. siRNA (d) or Ubc9 siRNA (e). Cells were labeled with [35S] methionine/cysteine for 4 hours. Cells were placed on ice and transferred to a 4°C cold room where they were lysed with ice cold TNE buffer for 30 minutes on ice followed by homogenization with a 25G needle. Lysates were clarified in a cooled micro centrifuge and then adjusted to 60% sucrose and overlaid with a discontinuous sucrose gradient and centrifuged at 100,000 X g at 4C for at least 18 hrs. Gradients were fractioned into 11 equal samples using a Biocomp piston fractionator and adjusted to 1x lysis buffer. Viral proteins were immunoprecipitated with patient serum, separated by SDS PAGE, and visualized by phosphorimaging using The Discovery Series Quantity One software. Lipid raft floatation experiments were carried out in triplicate. Representative over-exposed gels are shown so that viral proteins associated with lipids rafts can be more easily visualized. (f) Immunoblot of transfected cell lysates; untransfected (lane 1), NL4-3 + Ctr. siRNA (lane 2), NL4-3 + Ubc9 siRNA (lane 3). (g) Ratio of gp120/Pr55 proteins associated with lipid rafts. (h) Percent of total cellular Gag associated with lipid rafts.
