FIG. 3.
GM1 colocalizes with Gag and Env and is polarized to the VS. (A) JurkatLAI cells were stained with MAb 50-69 for surface-expressed Env (red) and with B-Ctx for GM1 (green), fixed, permeabilized with 0.1% Triton X-100, and incubated with rabbit antisera against HIV-1 Gagp17/p24 (blue) obtained from CFAR, United Kingdom, followed by anti-human-TRITC, streptavidin-FITC, and anti-rabbit-Cy5. Merging of the three colors illustrates colocalization, which is superimposed in white on the corresponding Nomarski image. (B) Conjugates between equal numbers of JurkatLAI effector cells (lower cell in conjugate) and freshly isolated resting primary CD4+ T target cells negatively enriched from normal PBMC (upper cell) were made on poly-l-lysine-treated coverslips for 1 h at 37°C, during which cells were stained for CD4 with L120 (blue; CFAR, United Kingdom) and for Env with 50-69 (red). Conjugate evolution was arresting by fixing with paraformaldehyde prior to staining for GM1 with B-Ctx (green), followed by anti-mouse-Cy5, anti-human-TRITC, and streptavidin-FITC. Merging of the three colors illustrates colocalization, which is superimposed in white on the corresponding Nomarski image. Arrows point to regions of GM1 and Env in the target cell membrane. (C) Conjugates between JurkatLAI effector and primary CD4+ T target cells were made as described above, during which cells were stained for Env with 50-69 (red). Conjugate evolution was arrested by fixing with paraformaldehyde prior to staining with B-Ctx (green). Conjugates were then permeabilized in 0.1% Triton X-100 and labeled with rabbit antisera against Gagp17/p24 (blue), followed by anti-human-TRITC, streptavidin-FITC, and anti-rabbit-Cy5. Merging of the three colors illustrates colocalization, which is superimposed in white on the corresponding Nomarski image.