Fig. 6: ABA does not activate B2 and B3 RAFs in plants.

a The phosphorylation of the conserved phosphosite in RAF2 and RAF3 showing enhanced phosphorylation by mannitol but not ABA treatment. The relative intensity of the phosphopeptide was obtained from previous phosphoproteomics results (n = 3 biological replicates). b Sequence alignment showing the conserved phosphosites (indicated by arrows) in the activation loop of Arabidopsis B2, B3, B4 RAFs, and PpARK/PpCTR1 from Physcomitrella patens. The conserved serine residues corresponding to Ser1029 in PpARK/PpCTR1 are highlighted by the red arrow. c Activation of the reporter gene by wild type and the non-phosphorylatable mutants of Ser763, Ser766, and Thr770 in RAF3 in transient reporter gene expression in protoplasts of OK¹⁰⁰-oct. RAF3^K636R (RAF3^KR), a kinase-dead form of RAF3, is used as a control. Error bars, SEM (n = 3 individual transfections). d Phosphorylation of SnRK2.6^KR by recombinant kinase domain of RAF3 and RAF10. Wild type and mutated recombinant GST-RAF3-KD (left panel) and GST-RAF10-KD (right panel) was used to determine the phosphorylation of SnRK2.6^KR expressed and purified from E. coli in the presence of [γ-³²P]ATP. GST-RAF3^K636R (RAF3^KR) and GST-RAF10^K515R (RAF10^KR) were used as negative controls. Autoradiograph (upper) and Coomassie staining (lower) show phosphorylation and loading, respectively, of purified GST-RAF3-KD, GST-RAF10-KD, and HIS-SnRK2.6^KR. Images shown are representative of at least two independent experiments. Source data are provided in Source Data.