Figure 5.
Figure 5. Lysosomal targeting of KRas. (A) COS-1 cells expressing GFP-KRas were treated with 100 ng/ml EGF-TRITC for 2 h. Triple labeling of GFP-KRas, LAMP1 (blue channel), and EGF-TRITC (red channel) is shown, and arrows indicate colocalization. (B and C) GFP-KRas–expressing cells were treated either with 20 nM B-A1 (B) or 25 µM leupeptin (C) and EGF. Immunolabeling with anti-LAMP1 antibody shows colocalization of KRas (green channel) with LAMP1 (red channel) on perinuclear ringlike structures (see arrows in insets of B and insets 1–3 of C); arrowheads (B, insets) point to KRas-positive vesicles not labeled with LAMP1. TO-PRO 3 is a nuclear marker (blue channel). (D) Quantification of LAMP1/GFP-KRas colocalization from experiments shown in A–C. Statistical significances of differences between different treatments were determined using the Student's t test. Data are means ± SEM; *, P < 0.05; ***, P < 0.001. (E and F) GFP-HRas–expressing cells treated with B-A1 (E) or leupeptin (F) and EGF show colocalization of HRas (green channel) with GMAP210 (Golgi marker; blue channel) but not with LAMP1 (red channel). (G) In vivo imaging of GFP-KRas–expressing cells after treatment with B-A1, leupeptin, and EGF reveals MVB-like structures containing intralumenal GFP-KRas–positive membranes (z1–z12). (H and H′) 3D tomographic reconstruction of confocal sections z1–z11 (H) and z1–z7 (H′). (I) GFP-Rab7–expressing cells treated with B-A1 and EGF and labeled with anti–pan-Ras exhibit considerable colocalization (arrows). Arrowheads point to Rab7-positive vesicles not labeled with the pan-Ras antibody. (J) HA-KRas stability was measured in control and B-A1 plus leupeptin–treated cells. Before stimulation with 100 ng/ml EGF, starved cells were preincubated for 6 h with 100 µg/ml CHX (control) or CHX in combination with B-A1 plus leupeptin (B-A1 + Leu). Equal amounts (15 µg) of cell lysates from 0, 3, 6, 15, and 21 h after EGF addition were electrophoresed, and the levels of HA-KRas detected with an anti-HA antibody in both control and B-A1 plus leupeptin–treated cells are shown (Western blot; n = 5). Actin was used as a loading control. Quantification analysis by densitometry was performed. Data are the mean ± SEM; *, P < 0.05; **, P < 0.01 (Student's t test). Bars: (A–C, E, F, and I), 20 µm; (G, H, and insets) 2 µm.

Lysosomal targeting of KRas. (A) COS-1 cells expressing GFP-KRas were treated with 100 ng/ml EGF-TRITC for 2 h. Triple labeling of GFP-KRas, LAMP1 (blue channel), and EGF-TRITC (red channel) is shown, and arrows indicate colocalization. (B and C) GFP-KRas–expressing cells were treated either with 20 nM B-A1 (B) or 25 µM leupeptin (C) and EGF. Immunolabeling with anti-LAMP1 antibody shows colocalization of KRas (green channel) with LAMP1 (red channel) on perinuclear ringlike structures (see arrows in insets of B and insets 1–3 of C); arrowheads (B, insets) point to KRas-positive vesicles not labeled with LAMP1. TO-PRO 3 is a nuclear marker (blue channel). (D) Quantification of LAMP1/GFP-KRas colocalization from experiments shown in A–C. Statistical significances of differences between different treatments were determined using the Student's t test. Data are means ± SEM; *, P < 0.05; ***, P < 0.001. (E and F) GFP-HRas–expressing cells treated with B-A1 (E) or leupeptin (F) and EGF show colocalization of HRas (green channel) with GMAP210 (Golgi marker; blue channel) but not with LAMP1 (red channel). (G) In vivo imaging of GFP-KRas–expressing cells after treatment with B-A1, leupeptin, and EGF reveals MVB-like structures containing intralumenal GFP-KRas–positive membranes (z1–z12). (H and H′) 3D tomographic reconstruction of confocal sections z1–z11 (H) and z1–z7 (H′). (I) GFP-Rab7–expressing cells treated with B-A1 and EGF and labeled with anti–pan-Ras exhibit considerable colocalization (arrows). Arrowheads point to Rab7-positive vesicles not labeled with the pan-Ras antibody. (J) HA-KRas stability was measured in control and B-A1 plus leupeptin–treated cells. Before stimulation with 100 ng/ml EGF, starved cells were preincubated for 6 h with 100 µg/ml CHX (control) or CHX in combination with B-A1 plus leupeptin (B-A1 + Leu). Equal amounts (15 µg) of cell lysates from 0, 3, 6, 15, and 21 h after EGF addition were electrophoresed, and the levels of HA-KRas detected with an anti-HA antibody in both control and B-A1 plus leupeptin–treated cells are shown (Western blot; n = 5). Actin was used as a loading control. Quantification analysis by densitometry was performed. Data are the mean ± SEM; *, P < 0.05; **, P < 0.01 (Student's t test). Bars: (A–C, E, F, and I), 20 µm; (G, H, and insets) 2 µm.

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