FIGURE 5.

Functional characterization of SH4 chimeric Nef.GFP proteins: down-regulation of cell surface CD4, Lck retargeting, and inhibition of F-actin ruffling. A, CD4 cell surface expression in Jurkat TAg cells transfected with expression constructs for GFP, Nef.GFP, or Fyn-NefΔSH4.GFP. Twenty-four hours post-transfection, cells were stained for surface expression with allophycocyanin (APC)-conjugated antibodies against hm CD4 and analyzed by flow cytometry. R1, GFP-negative cells; R2, high level GFP expressing cells. Red text, MFIs of cells stained for CD4. B, down-regulation activity of the various chimeric Nef.GFP proteins. CD4 surface levels of GFP transfected cells were set to 100%. The data are means ± S.D. of at least three independent experiments. C, representative confocal pictures of Jurkat TAg cells transfected with expression constructs for GFP, Nef.GFP, or Fyn-NefΔSH4.GFP. Cells were plated on poly-l-lysine-coated cover glasses, fixed, and stained. Shown is a merge of endogenous (end.) Lck (red) and GFP (green). Scale bar, 10 μm. D, quantification of the frequency of cells displaying pronounced intracellular Lck accumulation. The data represent average values from three independent experiments ± S.D. with at least 100 cells analyzed per condition. E, representative maximum projections of confocal Z stacks of Jurkat CCR7 T cells transfected with expression constructs for GFP, Nef.GFP, or Fyn-NefΔSH4.GFP. Cells were plated on poly-l-lysine-coated cover glasses, stimulated with hm SDF-1α, fixed, and stained for F-actin using phalloidin-TRITC. Shown is a merge of F-actin (red) and GFP (green). Scale bar, 10 μm. F, quantification of cells that show full inhibition of F-actin ruffling. The data represent average values from three independent experiments ± S.D. with at least 100 cells analyzed per condition.