Fig. 4.

a Time kinetic of UGD phosphorylation by AtMPK3. A phosphorylation assay was set up and aliquots were removed after 5, 10, 15, or 20 min. Upper row, undiluted sample; lower row 1:3 diluted sample. b Specificity of mAB UGD-P. Samples from a phosphorylation assay were spotted on a nitrocellulose membrane. In control assays (AtMPK3), the two kinases were heat inactivated before they were added to the phosphorylation assay. The UGD mAB recognizes both forms of UGD whereas the mAB UGD-P only interacts with phosphorylated UGD. c Western blot with mAB-UGD-P showing the specificity of the phosphorylation reaction. Only the full assay in the left lane leads to a strong western blot signal as predicted. Controls, in which either the MPK3 protein in missing (middle lane) or the kinase substrate ATP was left out (right lane), did not show any signal with the mAB-UGD-P specific antibody