YFP-H-RAS (N17S) mutant inhibits Shoc2 recruitment to endosomes.

<p><b>A</b>, HeLa cells were transfected with YFP-H-RAS (N17S), serum-starved and then incubated with 10 ng/ml EGF for 12 min at 37°C. Cells were then fixed, permeabilized and stained with anti- Shoc2 antibodies and secondary Cy3 donkey anti-rabbit. Insets show high magnification images of the regions of the cell indicated by the white rectangle. Cells expressing YFP-H-RAS (N17S) are outlined in the Shoc2 image. Scale bars, 10 µm. <b>B</b>, HeLa cells were transfected with the YFP-H-RAS (N17S) mutant and serum-starved. Cells were then incubated with 10 ng/ml EGF for 12 min at 37°C and lysed. The lysates were probed for Shoc2, RAS, phospho-ERK1/2 and total ERK1/2 by western blotting. <b>C</b>, Multiple images from the experiments exemplified in <b><i>A</i></b> were inspected, and the percentage of cells containing Shoc2 endosomes was calculated (+/−S.D.). The data are representative of 3 independent experiments, a vs. b, P<0.001 (one-way ANOVA test using SigmaStat 3.5 was used to determine differences).