FIGURE 3.

SmgGDS-607 binds directly and specifically to K-Ras CVIL, and SmgGDS-558 binds unprenylated WT K-Ras CVIM or CVIL in vitro. A, 100 ng of recombinant His-tagged SmgGDS-607 and SmgGDS-558 were allowed to interact with 250 ng of recombinant GST-Myc-tagged full-length K-Ras CVIM or K-Ras CVIL proteins prebound to glutathione-Sepharose 4B beads. The complexes were isolated by pelleting the beads and subjected to ECL-Western blotting using a SmgGDS antibody. The results are shown as the optical density of the SmgGDS protein signal in the immunoblots and are the means ± S.E. of three independent experiments. ns, not significant; **, p < 0.01 by Student's t test. B, Biotin-PEG₂-K-Ras CVIM or CVIL peptides with the sequence KKKKKKSKTKCVI(M/L) (top panel) were prebound (10 μg) to streptavidin beads and allowed to interact with HEK-293T cell lysates expressing either SmgGDS-607-HA, SmgGDS 558-HA, or HA vector. The complexes were isolated by pelleting the beads and subjected to ECL-Western blotting using HA antibody. The results are representative of three (30 sec. exposure) or two (Overnight exposure) independent experiments. C, lysates of HEK-293T cells expressing SmgGDS-607-HA were prepared as increasing concentrations of total cellular protein (45 μg to 2250 μg) (top left panel) or 1000 μg of total cellular protein (top right panel) and allowed to interact with the biotin-K-Ras CVIL peptide (10 μg) that was prebound to streptavidin beads. Full-length recombinant GST-K-Ras-CVIM or -CVIL proteins were added at increasing concentrations (0.1–1 μg) to the reaction mixtures (top right panel). SmgGDS-607-HA that was bound to the biotin-K-Ras CVIL peptide was isolated by pelleting the streptavidin beads and then subjected to ECL-Western blotting using HA antibody. The results are representative of two (top left panel) or three (top right panel) independent experiments. The graph shows the optical density (means ± S.E.) of the immunoblotted SmgGDS-607-HA protein that was pulled down by the biotin-K-Ras CVIL peptide in the presence of the indicated concentrations of full-length recombinant GST-K-Ras-CVIM or -CVIL proteins. *, p < 0.05 by one-way analysis of variance with Dunnett's post-test compared with the control value from the sample that did not receive full-length recombinant GST-K-Ras protein.