Click on image to view larger version.



Fig. 1. Substitution of the CAAX motif of RhoA(63L) with the CVLS motif from farnesylated H-Ras results in a protein that is not altered in subcellular location but is sensitive to FTase, but not GGTaseI, inhibitor treatment. A, lysates from NIH 3T3 cells stably or transiently expressing HA epitope-tagged RhoA(63L) proteins were normalized for total protein. The proteins were resolved by SDS-PAGE, and expression was determined by Western blot analysis using anti-HA epitope antibody. B, NIH 3T3 cells transiently expressing the indicated GFP-tagged RhoA(63L) proteins were cultured in growth medium supplemented with vehicle (DMSO), FTI-2153, or GGTI-2166 were analyzed as live cells. Cells were then visualized using a Axioskop 2 microscope and Openlab digital imaging software. C, alternatively prenylated, but not unprenylated, shows a similar subcellular location as geranylgeranylated RhoA(63L). NIH 3T3 cells stably expressing HA epitope-tagged RhoA(63L)-WT, RhoA(63L)-CVLS, and RhoA(63L)-SLVL were fixed, and the proteins were visualized by indirect immunofluorescence analyses using anti-HA epitope antibody and a FITC-conjugated antimouse secondary antibody. Data shown are representative of three independent experiments.