Figure 7.
Figure 7. Grb2 and Raf1 are recruited to KRas-positive LEs. (A and B) COS-1 cells were transfected with Grb2-YFP and treated with B-A1 for 6 h. (A) Starved cells show diffuse cytosolic distribution of Grb2-YFP. (B) After 45 min of EGF-TRITC stimulation, Grb2 is redistributed onto perinuclear vesicles. Colocalization of EGF-TRITC (red channel), immunolabeled EGFR (blue channel), and Grb2 (green channel) is observed (inset). (C and D) Grb2-YFP and CFP-KRas were coexpressed in COS-1 cells, and then cells were treated with B-A1. (C) Control cells show diffuse cytoplasmic distribution of Grb2-YFP. Insets show colocalization of immunolabeled EGFR and CFP-KRas but not Grb2-YFP on both endosomes (arrows) and the PM (arrowheads). (D) After 45 min of EGF-TRITC stimulation, Grb2 is redistributed onto vesicular structures. Colocalization of EGF (red channel), immunolabeled EGFR (gray channel), Grb2 (green channel), and KRas (blue channel) is observed (arrows). (E and F) COS-1 cells were transfected with YFP-Raf1 and treated with B-A1 for 6 h. (E) Starved cells show diffuse cytoplasmic distribution of YFP-Raf1. (F) After 45 min of EGF-TRITC stimulation, Raf1 was recruited, to some extent, to vesicular structures (inset). Colocalization of internalized EGF (red channel) and Raf1 (green channel) could be observed (inset). (G and H) YFP-Raf1 and CFP-KRas were coexpressed in COS-1 cells, and then cells were treated with B-A1. (G) Control cells show a diffuse cytosolic pattern of YFP-Raf1. Some degree of colocalization of YFP-Raf1 (green channel) and CFP-KRas (blue channel) can be observed (white arrows in the merge inset and red arrows in the color channel insets). (H) After 45 min of EGF-TRITC stimulation, Raf1 is redistributed onto vesicular structures. Clear colocalization of endocytosed EGF (red channel), Raf1 (green channel), and KRas (blue channel) is observed (white arrows in the merge inset and red arrows in the color channel insets). (B–D and F–H) Dotted boxes define the areas from which the corresponding insets were generated. Bars, 20 µm.

Grb2 and Raf1 are recruited to KRas-positive LEs. (A and B) COS-1 cells were transfected with Grb2-YFP and treated with B-A1 for 6 h. (A) Starved cells show diffuse cytosolic distribution of Grb2-YFP. (B) After 45 min of EGF-TRITC stimulation, Grb2 is redistributed onto perinuclear vesicles. Colocalization of EGF-TRITC (red channel), immunolabeled EGFR (blue channel), and Grb2 (green channel) is observed (inset). (C and D) Grb2-YFP and CFP-KRas were coexpressed in COS-1 cells, and then cells were treated with B-A1. (C) Control cells show diffuse cytoplasmic distribution of Grb2-YFP. Insets show colocalization of immunolabeled EGFR and CFP-KRas but not Grb2-YFP on both endosomes (arrows) and the PM (arrowheads). (D) After 45 min of EGF-TRITC stimulation, Grb2 is redistributed onto vesicular structures. Colocalization of EGF (red channel), immunolabeled EGFR (gray channel), Grb2 (green channel), and KRas (blue channel) is observed (arrows). (E and F) COS-1 cells were transfected with YFP-Raf1 and treated with B-A1 for 6 h. (E) Starved cells show diffuse cytoplasmic distribution of YFP-Raf1. (F) After 45 min of EGF-TRITC stimulation, Raf1 was recruited, to some extent, to vesicular structures (inset). Colocalization of internalized EGF (red channel) and Raf1 (green channel) could be observed (inset). (G and H) YFP-Raf1 and CFP-KRas were coexpressed in COS-1 cells, and then cells were treated with B-A1. (G) Control cells show a diffuse cytosolic pattern of YFP-Raf1. Some degree of colocalization of YFP-Raf1 (green channel) and CFP-KRas (blue channel) can be observed (white arrows in the merge inset and red arrows in the color channel insets). (H) After 45 min of EGF-TRITC stimulation, Raf1 is redistributed onto vesicular structures. Clear colocalization of endocytosed EGF (red channel), Raf1 (green channel), and KRas (blue channel) is observed (white arrows in the merge inset and red arrows in the color channel insets). (B–D and F–H) Dotted boxes define the areas from which the corresponding insets were generated. Bars, 20 µm.

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