Pull down assay of RhoA.

<p>(A) A RhoA bead pull down assay revealed lowered levels of activated RhoA in Ibuprofen treated cells. Treatment with downstream Rho kinase inhibitor Y-27632 showed no difference in RhoA level compared to control. After treatment with lysophosphatidic acid, significantly more phosphorylated RhoA could be detected in the Western Blot. Treatment with downstream Rho kinase inhibitor Fasudil showed no difference in RhoA level compared to control. Cell lysates were blotted and stained with RhoA-specific monoclonal antibody. Staining is visualized with horseradish peroxidase and the 3, 3′-diaminobenzidine substrate. Molecular weight of stained protein bands corresponds to about 20 kDa as reported for RhoA. (B) Relative RhoA level was decreased to approximately 50% of control after Ibuprofen treatment whereas RhoA level after LPA treatment was increased to 200% of control. Data are normalized grey values ± SEM of 4 independent experiments.