RhoGTP pull down assay and in situ RhoGTP affinity assay. RhoGTP pull down assay showing (A) constitutive and (B) LPA induced and control levels of active RhoGTP in Dlc1wt/wt, Dlc1wt/gt and Dlc1gt/gt SFME cell lines. (C) Dlc1 SFME cells exponentially growing on glass slides were fixed and cells were incubated with 50 μg/mL of soluble GST (i) or GST-RBD (ii). RhoGTP-bound RBD was detected by GST immunostaining. (D) Plot showing the RhoGTP to total Rho protein by scanning the relative intensity of the protein bands in non-treated and LPA induced in SEME cell lines. Means and standard error of mean determined from at least six independent experiments using cell lines derived from different mouse embryos are given. *P < 0.001, by two way ANOVA test.