Fig. 5. Stable N-RAS palmitoylation requires acidic linker residues. N-RAS constructs exhibit equivalent de-palmitoylation kinetics, with t_1/2=12-15 minutes (A). Steady-state [³H]-palmitate labelling is equivalent for N-RAS constructs (B) but significantly reduced when acidic residues are lost compared with control (C; HVR-N-DA, HVR-N-Q-2-ala and HVR-N-ala versus HVR-N). Representative blots of [³H]-palmitate labelling are displayed, with re-probed GFP blots used for normalisation; the graphs incorporate the means of the [³H]-palmitate signal corrected for GFP expression and normalised to N-RAS labelling (B,C). The percentage total protein in the P100 fraction reveals equivalent levels of strong membrane binding among the constructs (D). The graphs display means from 3-5 independent experiments.