Quercetin binding blocks PID kinase activity. Quercetin binding blocks PID kinase activity. (A, B) In vitro autophosphorylation of PID‐GST is inhibited by quercetin and chelerythrine while IAA and NPA have only slight effects. Coomassie stains (left panels) of non‐phosphorylated PID (PID) was used as loading control (A). Autoradiographies (right panels) of autophosphorylated PID (PID‐P), represented by the upper band in the Coomassie stain (Christensen et al, 2000), were quantified (B) and signal intensities were plotted against solvent controls (C; lower panel; means±s.e.; n=3). Significant differences (unpaired t‐test with Welch's correction, P<0.05) to solvent controls are indicated by asterisks. (C) PID‐GST binds specifically quercetin and to a lesser amount as well IAA (mean±s.e.; n=4). Background drug binding to column material or GST alone (background) was corrected by subtracting specific binding to column‐bound GST from column‐bound PID‐GST. (D) Microsomes prepared from PID loss‐ and gain‐of‐function lines show reduced and enhanced specific quercetin binding, respectively, but reciprocal specific NPA binding (mean±s.d.; n=4). Note that material was prepared from heterozygous pid (pid+/−) plants since, due to technical limitations, a determination of homozygosity by shoot phenotyping or genotyping was not possible. Reported values (C, D) are the means of specific radiolabelled drug bound in the absence of cold drug (total) minus radiolabelled drug bound in the presence of cold drug (unspecific). Significant differences (unpaired t‐test with Welch's correction, P<0.05) between GST alone (background; A) or wild‐type and mutant microsomes (B) are indicated by asterisks. Figure source data can be found with the Supplementary data. Sina Henrichs et al. EMBO J. 2012;31:2965-2980 © as stated in the article, figure or figure legend