The D287A mutation of BAK1 blocks its cleavage. A, Scheme of BAK1 protein domains and sites for mutagenesis. B, VASD residues are required for BAK1 cleavage. Arabidopsis protoplasts expressing HA-tagged BAK1 mutants, in which the indicated four amino acids were mutagenized to Ala, were analyzed by western blot (WB)with an α-HA antibody. C, D287 residue is required for BAK1 cleavage in Arabidopsis protoplasts. D, D287 residue is required for BAK1 cleavage in N. benthamiana. The N. benthamiana leaves transiently expressing HA-tagged BAK1 or BAK1D287A mutant were analyzed by WB with α-HA antibody. E, D287 residue is required for BAK1 cleavage in Arabidopsis 35S::BAK1-HA transgenic plants. F, D287 residue is required for BAK1 cleavage in Arabidopsis pBAK1::BAK1-GFP transgenic plants. G, D287E mutation blocks BAK1 cleavage in Arabidopsis protoplasts. H, D287 residue of BAK1 is conserved among Arabidopsis SERKs. I, BAK1 D287 corresponding residue in other SERK members is required for cleavage in Arabidopsis protoplasts. The above experiments were repeated three times with similar results.