(A) Gal-1-complexation FRET analysis of K-ras mutants and its parent construct transiently expressed in BHK cells. (B) RBD-recruitment FRET analysis of K-ras mutants and its parent construct transiently expressed in BHK cells with (+) or without (−) 5 μM compactin treatment for 48 hr. Compactin treatment relocalized K-ras mutant/RBD complexes to the cytoplasm, allowing us to assess binding in solution. (A, B) Numbers in bars give number of analyzed cells from three independent experiments. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant, ***p < 0.001). (C) RBD-pulldown experiment quantification of the active, GTP-bound forms of wt and mutant K-ras. +EGF denotes stimulation with 100 ng/ml EGF. −EGF serum-starved cells. +GAP incubation with GAP domain of NF1, to assay for GAP sensitivity. The graphs represent the averages of active K-ras mutants normalized to wt K-ras + EGF-stimulation from three independent experiments. Blue vertical line annotates the activity of wt K-ras when stimulated with EGF. Error bars represent the standard error of the mean (±SEM). Statistical analysis was performed as described in ‘Materials and methods’ (NS, non-significant, ***p < 0.001). (D) Western blot analysis of phosphorylated MEK, ERK, and AKT and total MEK, ERK, and AKT in BHK cells transiently expressing EGFP only, mCFP-K-ras-wt, mCFP-K-ras-V152G, or mCFP-K-ras-R164Q. Cells were serum starved and collected without EGF stimulation (−EGF) or after stimulation with 100 ng/ml EGF for 10 min (+EGF). Equal expression of Ras constructs can be seen in the GFP row, equal loading in the β-actin row. (E) Validation of comparable protein expression levels of HA-tagged K-ras mutants stably expressed in NIH/3T3 cells. Anti-HA-tag Western blot.