FIGURE 2.
K-Ras CVIM is farnesylated, whereas K-Ras CVIL is geranylgeranylated, and SmgGDS-607 recognizes the last amino acid in the CAAX motif for binding. A, SmgGDS-607-HA was expressed in HEK-293T cells with Myc-tagged WT K-Ras CVIM, CVIL, or their nonprenylatable mutants K-Ras SVIM and K-Ras SVIL, or a Myc vector. 90 min post-transfection, the cells were treated with either GGTI (15 μm) or vehicle. After 24 h, cells were lysed, and an aliquot of each lysate was subjected to ECL-Western blotting using Myc antibody (Cell Lysates). The remaining volume of each cell lysate was immunoprecipitated with HA antibody, and the immunoprecipitates were immunoblotted using HA and Myc antibodies (HA Immunoprecipitates). The results are shown as the optical density of IP Myc-K-Ras divided by the optical density of IP SmgGDS-HA and are the means ± S.E. of three independent experiments. *, p < 0.01 by one-way analysis of variance with Dunnett's post-test compared with the control value of Myc-K-Ras CVIM pulled down (lane 1). B, HEK-293T cells were transfected with a cDNA encoding either Myc-tagged WT K-Ras CVIM (top panel) or mutant K-Ras CVIL (bottom panel). 90 min post-transfection, cells were treated with either FTI-277 (10 μm), GGTI-298 (10 μm), both FTI and GGTI, mevastatin (10 μm), or vehicle control. After 24 h, the cells were lysed, and an aliquot of each lysate was subjected to ECL-Western blotting using Myc antibody (Total Cell Lysate). The remaining volume of each cell lysate was subjected to Triton X-114 fractionation, and equal volumes of the aqueous phase, detergent phase, or total cell lysate were immunoblotted using Myc antibody, antibody to SmgGDS (aqueous phase marker), or antibody to Gβ1 subunit (detergent phase marker).