Figure 3.

ERL1–TMM complexes function as receptors of EPF1 and EPF2 in vitro. (A) ERfs alone fail to interact with EPF1 or EPF2 in pull-down assays. The 6xHis-tagged ER^LRR, ERL1^LRR, and ERL2^LRR were incubated individually with EPF1 or EPF2, and the mixtures were then flowed through Ni-NTA beads. After extensive washing, the bound proteins were eluted and analyzed with SDS-PAGE followed by Coomassie blue staining. (B) EPF1 directly binds to the ERL1^LRR–TMM^LRR complex. (Left panel) Size exclusion chromatography analysis of the interaction between EPF1 and ERL1^LRR–TMM^LRR; the elution volumes and the molecular weight markers are indicated at the top. (Right panel) SDS-PAGE analysis of peak fractions from the left panel. (C) Quantification of the binding affinity of EPF1 (left) or EPF2 (right) to ERL1^LRR or ERL1^LRR–TMM^LRR by ITC. (nd) No detectable binding. EPF1 or EPF2 was titrated into ERL1^LRR or ERL1^LRR–TMM^LRR protein in the ITC cell. Integrated heat measurements from ITC are shown. The calculated stoichiometry (N) and the dissociation constant (K_d) are indicated.