Figure 1. EGF induces KRas translocation... | Rockefeller University Press

Figure 1.

$Figure 1. EGF induces KRas translocation to the early endosomal compartment. COS-1 cells were transiently transfected with GFP-KRas, and its intracellular localization was analyzed by confocal microscopy. (A) Colocalization of GFP-KRas and EEA1 on intracellular structures. (B) Cells treated with 100 ng/ml EGF-TRITC for 20 min show increased colocalization between GFP-KRas, EEA1, and EGF-TRITC. (C) Quantification of cells in B. Statistical significances of differences between control and EGF treatment were determined using the Student's t test. Data are means ± SEM; **, P < 0.01. (D) A more detailed confocal image of early endosomal structures positive for GFP-KRas, EEA1, and EGF-TRITC is shown. Red dotted circles indicate the perimeter of magnified endosomes. (E) Likewise, in EGF-stimulated cells, endocytosed EGFR detected by immunofluorescence colocalizes with GFP-KRas and EGF-TRITC. (F) NIH3T3-Wt8 cells treated with 100 ng/ml EGF-TRITC for 20 min also confirmed colocalization of GFP-KRas, EEA1, and EGF-TRITC. (G) In NIH3T3 cells, a significant increase of GFP-KRas together with EGFR is detected in endosomal fractions isolated by sucrose density gradients after EGF treatment (Western blot), and the results presented are representative of three independent experiments (graph). (H) Similarly, after EGF stimulation, subcellular fractionation by sucrose gradients using nontransfected NIH3T3-Wt8 cells shows enrichment of endogenous KRas and EGFR in endosome fractions (Western blot). Colocalization is indicated with white (merge) or red (separate channels) arrows. (A, B, E, and F) Dotted boxes define the areas from which the corresponding insets were generated. Bars: (A, B, E, and F) 20 µm; (D) 500 nm.$

EGF induces KRas translocation to the early endosomal compartment. COS-1 cells were transiently transfected with GFP-KRas, and its intracellular localization was analyzed by confocal microscopy. (A) Colocalization of GFP-KRas and EEA1 on intracellular structures. (B) Cells treated with 100 ng/ml EGF-TRITC for 20 min show increased colocalization between GFP-KRas, EEA1, and EGF-TRITC. (C) Quantification of cells in B. Statistical significances of differences between control and EGF treatment were determined using the Student's t test. Data are means ± SEM; **, P < 0.01. (D) A more detailed confocal image of early endosomal structures positive for GFP-KRas, EEA1, and EGF-TRITC is shown. Red dotted circles indicate the perimeter of magnified endosomes. (E) Likewise, in EGF-stimulated cells, endocytosed EGFR detected by immunofluorescence colocalizes with GFP-KRas and EGF-TRITC. (F) NIH3T3-Wt8 cells treated with 100 ng/ml EGF-TRITC for 20 min also confirmed colocalization of GFP-KRas, EEA1, and EGF-TRITC. (G) In NIH3T3 cells, a significant increase of GFP-KRas together with EGFR is detected in endosomal fractions isolated by sucrose density gradients after EGF treatment (Western blot), and the results presented are representative of three independent experiments (graph). (H) Similarly, after EGF stimulation, subcellular fractionation by sucrose gradients using nontransfected NIH3T3-Wt8 cells shows enrichment of endogenous KRas and EGFR in endosome fractions (Western blot). Colocalization is indicated with white (merge) or red (separate channels) arrows. (A, B, E, and F) Dotted boxes define the areas from which the corresponding insets were generated. Bars: (A, B, E, and F) 20 µm; (D) 500 nm.