Figure 7
Figure 7. Syntenin binds multiple IL-5Rα chains. (A) Schematic representation of the experimental design of the PCA. Venus, an improved version of yellow fluorescent protein (YFP), was fragmented into nonfunctional parts that were fused to the N-termini of the intracellular domains of IL-5Rα (YFP1–IL-5Rα and YFP2–IL-5Rα). When both of the fusion products bind syntenin, the nonfunctional Venus parts regain fluorescence. (i) As a control, expression of the fusion products with a single PDZ domain of syntenin should not result in functional restoration of the Venus protein. (ii) As an additional control, we fused YFP-1 to the C-terminus of IL-5Rα (IL-5Rα–YFP1) instead of its N-terminus. Because syntenin binds the C-terminus of IL-5Rα, no additional fluorescence is expected upon full-length syntenin or PDZ2 cotransfection (iii and iv, respectively). (B) HEK293 cells were transiently transfected with YFP1–IL-5Rα and YFP2–IL-5Rα together with empty vector, or syntenin's second PDZ domain (PDZ2), or full-length syntenin (left panel). The percentage of positive cells was determined by flow cytometry. At right, similar conditions were assayed but an IL-5Rα–YFP1 instead of YFP1–IL-5Rα fusion product was used. Empty vector transfections per experiments were set at 100%. Data depict 3 independent experiments (± SD). *P < .05. (C) YFP1–IL-5Rα and YFP2–IL-5Rα dose-dependent increase of fluorescence in the presence of full-length syntenin. Cells were transfected with fixed amounts of empty vector, syntenin PDZ2 or full-length syntenin (each 0.5 μg), and increasing concentrations of YFP1–IL-5Rα and YFP2–IL-5Rα. Cells were analyzed 48 hours after transfection for YFP fluorescence by flow cytometry. Percentage of YFP-fluorescent cells is indicated. One representative example of 2 identical experiments is indicated. (D) Representative dot plots of cells transfected with empty vector, syntenin PDZ2 or full-length syntenin (0.5 μg), together with 0.5 μg of YFP1–IL-5Rα and YFP2–IL-5Rα. Forward scatter is plotted against YFP fluorescence in the FL-1 channel. Geometric mean fluorescent intensities of the positive cells are indicated in the squares. (E) Immunoprecipitation (IP) of syntenin–IL-5Rα complexes from transfected HEK293 cells in the presence of the chemical cross-linker DSP. The left panels indicate expression of the myc–IL-5Rα (17.5 kDa) and HA-tagged syntenin (35 kDa) in whole-cell lysates (WCLs). The middle panel shows HA immunoprecipitates analyzed by nonreducing SDS-PAGE and myc-immunoblotting (IB), whereas the right panel shows HA immunoreactivity of the same samples blotted in duplicate.

Syntenin binds multiple IL-5Rα chains. (A) Schematic representation of the experimental design of the PCA. Venus, an improved version of yellow fluorescent protein (YFP), was fragmented into nonfunctional parts that were fused to the N-termini of the intracellular domains of IL-5Rα (YFP1–IL-5Rα and YFP2–IL-5Rα). When both of the fusion products bind syntenin, the nonfunctional Venus parts regain fluorescence. (i) As a control, expression of the fusion products with a single PDZ domain of syntenin should not result in functional restoration of the Venus protein. (ii) As an additional control, we fused YFP-1 to the C-terminus of IL-5Rα (IL-5Rα–YFP1) instead of its N-terminus. Because syntenin binds the C-terminus of IL-5Rα, no additional fluorescence is expected upon full-length syntenin or PDZ2 cotransfection (iii and iv, respectively). (B) HEK293 cells were transiently transfected with YFP1–IL-5Rα and YFP2–IL-5Rα together with empty vector, or syntenin's second PDZ domain (PDZ2), or full-length syntenin (left panel). The percentage of positive cells was determined by flow cytometry. At right, similar conditions were assayed but an IL-5Rα–YFP1 instead of YFP1–IL-5Rα fusion product was used. Empty vector transfections per experiments were set at 100%. Data depict 3 independent experiments (± SD). *P < .05. (C) YFP1–IL-5Rα and YFP2–IL-5Rα dose-dependent increase of fluorescence in the presence of full-length syntenin. Cells were transfected with fixed amounts of empty vector, syntenin PDZ2 or full-length syntenin (each 0.5 μg), and increasing concentrations of YFP1–IL-5Rα and YFP2–IL-5Rα. Cells were analyzed 48 hours after transfection for YFP fluorescence by flow cytometry. Percentage of YFP-fluorescent cells is indicated. One representative example of 2 identical experiments is indicated. (D) Representative dot plots of cells transfected with empty vector, syntenin PDZ2 or full-length syntenin (0.5 μg), together with 0.5 μg of YFP1–IL-5Rα and YFP2–IL-5Rα. Forward scatter is plotted against YFP fluorescence in the FL-1 channel. Geometric mean fluorescent intensities of the positive cells are indicated in the squares. (E) Immunoprecipitation (IP) of syntenin–IL-5Rα complexes from transfected HEK293 cells in the presence of the chemical cross-linker DSP. The left panels indicate expression of the myc–IL-5Rα (17.5 kDa) and HA-tagged syntenin (35 kDa) in whole-cell lysates (WCLs). The middle panel shows HA immunoprecipitates analyzed by nonreducing SDS-PAGE and myc-immunoblotting (IB), whereas the right panel shows HA immunoreactivity of the same samples blotted in duplicate.

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