Phosphorylation of RhoA on ⁸⁸S and ¹⁰⁰T by active ERK1 <i>in vitro</i>.

<p><b>(A)</b> Phosphorylation of His-RhoA, His-Cdc42, and His-Rac1 by ERK1 <i>in vitro</i>. The phosphorylation of purified His-tagged Rho proteins by purified active ERK1 was performed with an <i>in vitro</i> ERK kinase assay kit in the presence of [γ-³²P]ATP as described in the Materials and Methods. ³²P was detected by autoradiography. <b>(B)</b> Quantification of the data from three independent experiments as described in (A). The intensity of the bands of ³²P was normalized against the intensity of the His-tagged protein loading. The error bar is standard error. * indicates p<0.05. <b>(C)</b> Phosphorylation of GST-RhoA and mutant proteins by purified ERK1 <i>in vitro</i>. The phosphorylation of GST-RhoA (5 μg) and various mutant GST-RhoA (5 μg) by purified active ERK1 was performed as described in (A). GST was used as a negative control. GST fusion protein loading was verified by Amido Black stain of the PVDF membrane. <b>(D)</b> Quantification of the data from three independent experiments as described in panel C. The intensity of the bands of ³²P was normalized against the intensity of the GST fusion protein loading. The error bar is standard error. * indicates p<0.05 and ** indicates p<0.01.