FIGURE 3.

CES1 impacts methylation status of RhoA. A, inhibition of CES1 specifically affects methylation status of RhoA. MDA-MB-231 cells were treated with siRNA or mock as described under “Experimental Procedures,” harvested, and processed for two-dimensional gel analysis of RhoA. A representative immunoblot is depicted showing unmethylated (U) and methylated (M) RhoA. B, quantitative representation of unmethylated RhoA from A expressed as a fraction of total RhoA. Data represent the mean ± S.E. of three independent experiments for luciferase (LUC) and CES1 and two independent experiments for CES2. C, CES1 demethylates RhoA in vitro. Lysates from MDA-MB-231 cells were prepared and incubated with purified CES1 enzyme as described under “Experimental Procedures.” A representative immunoblot is depicted showing unmethylated (U) and methylated (M) RhoA. D, quantitative representation of unmethylated RhoA from A expressed as a fraction of total RhoA. Data represent the mean ± S.E. of two independent experiments.