American Society of Hematology

Figure 4

$Figure 4. Nef TGN-association of active N-Ras downstream of Lck. (A) Representative confocal micrographs of Jurkat T lymphocytes expressing CFP.N-Ras (blue) together with the indicated RFP fusion proteins (red). Arrows indicate discrete or less compact localization of CFP.N-Ras in the presence of RFP/AxxA.RFP (1 arrow) or Nef.RFP (2 arrows), respectively. Scale bar indicates 10 μm. (B) Frequencies of cells that display dispersion of N-Ras into the expanded cytoplasmic pattern shown in the presence of Nef in panel A. Where indicated, Lck inhibitor (1μM) was added 3 hours before the analysis. Values are the arithmetic means of at least 3 independent experiments ± SD in which more than 100 cells were counted per condition. (C) Representative confocal micrographs of Jurkat T lymphocytes expressing HA.N-Ras (not shown) with the indicated RFP fusion proteins (red) and the Ras-binding domain of cRaf-1 fused to GFP as biosensor of Ras activity (RBD.GFP, green). Note that expression of Nef leads to a marked enrichment of active Ras at the TGN (indicated by the arrow). Scale bar indicates 10 μm. (D) Frequencies of cells that display accumulation of RBD.GFP at the TGN as shown in the presence of Nef in panel C. Values are the arithmetic means of at least 3 independent experiments ± SD in which more than 100 cells were counted per condition. Where indicated, Lck inhibitor (1μM) was added 3 hours before the analysis. (E) Membrane flotation analysis of Jurkat T lymphocytes expressing Lck.GFP with RFP or Nef.RFP. Lck.GFP and endogenous Lck were detected by an anti–Lck Ab. M indicates the membrane fraction; S, soluble fraction. TfR and MLC were used as markers for the membrane and soluble fractions, respectively. All samples were generated within the same experiment and run on the identical gel, but are separated by a vertical line to illustrate that lanes in between those shown were removed. (F) Subcellular fractionation. Postnuclear supernatants of homogenates of Jurkat T lymphocytes that were lentivirally transduced for expression of GFP or Nef.GFP (transduction efficiency above 98%) were subjected to sucrose density gradient centrifugation. Ten fractions were collected from the top of the gradient and analyzed by Western blotting for distribution of endogenous Lck, p-Lck394, p-Lck505, and p-Fyn409. For detection of N-Ras, Jurkat T lymphocytes were transfected with expression plasmids for HA.N-Ras and GFP or Nef.GFP plasmids. TGN46, TfR, and PLSCR1 serve as markers for gradient fractions containing TGN, RE, and PM, respectively.$

Nef TGN-association of active N-Ras downstream of Lck. (A) Representative confocal micrographs of Jurkat T lymphocytes expressing CFP.N-Ras (blue) together with the indicated RFP fusion proteins (red). Arrows indicate discrete or less compact localization of CFP.N-Ras in the presence of RFP/AxxA.RFP (1 arrow) or Nef.RFP (2 arrows), respectively. Scale bar indicates 10 μm. (B) Frequencies of cells that display dispersion of N-Ras into the expanded cytoplasmic pattern shown in the presence of Nef in panel A. Where indicated, Lck inhibitor (1μM) was added 3 hours before the analysis. Values are the arithmetic means of at least 3 independent experiments ± SD in which more than 100 cells were counted per condition. (C) Representative confocal micrographs of Jurkat T lymphocytes expressing HA.N-Ras (not shown) with the indicated RFP fusion proteins (red) and the Ras-binding domain of cRaf-1 fused to GFP as biosensor of Ras activity (RBD.GFP, green). Note that expression of Nef leads to a marked enrichment of active Ras at the TGN (indicated by the arrow). Scale bar indicates 10 μm. (D) Frequencies of cells that display accumulation of RBD.GFP at the TGN as shown in the presence of Nef in panel C. Values are the arithmetic means of at least 3 independent experiments ± SD in which more than 100 cells were counted per condition. Where indicated, Lck inhibitor (1μM) was added 3 hours before the analysis. (E) Membrane flotation analysis of Jurkat T lymphocytes expressing Lck.GFP with RFP or Nef.RFP. Lck.GFP and endogenous Lck were detected by an anti–Lck Ab. M indicates the membrane fraction; S, soluble fraction. TfR and MLC were used as markers for the membrane and soluble fractions, respectively. All samples were generated within the same experiment and run on the identical gel, but are separated by a vertical line to illustrate that lanes in between those shown were removed. (F) Subcellular fractionation. Postnuclear supernatants of homogenates of Jurkat T lymphocytes that were lentivirally transduced for expression of GFP or Nef.GFP (transduction efficiency above 98%) were subjected to sucrose density gradient centrifugation. Ten fractions were collected from the top of the gradient and analyzed by Western blotting for distribution of endogenous Lck, p-Lck394, p-Lck505, and p-Fyn409. For detection of N-Ras, Jurkat T lymphocytes were transfected with expression plasmids for HA.N-Ras and GFP or Nef.GFP plasmids. TGN46, TfR, and PLSCR1 serve as markers for gradient fractions containing TGN, RE, and PM, respectively.

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