Fig. 1.

AtPRK6^LRR specifically interacts with AtLURE1.2 in vitro. a AtLURE1.2 interacts with AtPRK6^LRR in a pull-down assay. The purified AtPRK^LRR and MIK1^LRR, MIK2^LRR proteins with 6 × His at the C-terminus bound to Ni-NTA were individually incubated with an excess of AtLURE1.2 protein. After extensive washing, the bound proteins were eluted and visualized by Coomassie blue staining following by SDS-PAGE. b AtLURE1.2 binding does not alter the monomeric state of AtPRK6^LRR in solution. Upper panel: gel filtration profiles of AtPRK6^LRR, AtLURE1.2, and AtPRK6^LRR-AtLURE1.2 complex. The black arrows indicate the molecular weights around the AtPRK6^LRR-AtLURE1.2 complex and AtLURE1.2. A₂₈₀ (mAU), micro-ultraviolet absorbance at the wavelength of 280 nm. Lower panel: coomassie blue staining of the peak fractions shown on the top following SDS-PAGE. M, molecular weight ladder (kDa). Numbers on top of SDS-PAGE panel indicate elution volumes. c Measurement of the binding affinity between AtPRK6^LRR and AtLURE1.2 by ITC. Left upper panel: twenty injections of AtLURE1.2 solution were titrated into AtPRK6^LRR solution in the ITC cell. The area of each injection peak corresponds to the total heat released for that injection. Left lower panel: the binding isotherm for AtPRK6^LRR-AtLURE1.2 interaction. The integrated heat is plotted against the molar ratio between AtLURE1.2 and AtPRK6^LRR. Data fitting revealed a binding affinity of about 3.1 μM. Right panel: binding affinity between AtPRK3^LRR and AtLURE1.2 by ITC