FIGURE 4.

LPA-stimulated MRK activation is inhibited by Rho proteins down-regulation. LPA stimulates activation of RhoA and RhoC proteins. U2OS cells (A) or EMDI cells (B) were treated with 25 μm LPA for the indicated times and then tested for activation of Rho proteins using pull-down assay with GST-Rhotekin-RBD. The last 3 lanes of each blot in A are pull-downs from steady-state lysates of cells transfected with the individual siRNAs that were used to control for band specificity. Each set of experiments was repeated 3–5 times; bands were quantified with the ImageJ program and expressed as means + S.E. in the corresponding histograms. C, UMDI cells, or D, EMDI cells, were transfected with control luciferase, RhoC or RhoA targeting siRNAs and then treated with 25 μm LPA for 5 min. Cell lysates were immunoblotted with the pMRK specific antibody to test for MRK activity, and with the indicated antibodies for controls. The pMRK bands were quantified with ImageJ. Histograms are means + S.E. of three independent experiments. p values representing Student's t test were *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001.