Figure 9 |. Analysis of phosphosite-specific kinase activities of BAK1.

a, Specific detection of BAK1-Y403 phosphorylation on affinity-purified recombinant BAK1^CD WT but not Y403F or kinase-dead (D416N) proteins, using α-pY403 antibodies and following an in vitro kinase assay with cold ATP. Blots were probed with α -BAK1 antibodies for loading control. b, Detection of BAK1-Y403, -S612, -threonine (T) and -tyrosine (Y) phosphorylation using phosphorylation specific antibodies on affinity-purified recombinant BAK1^CD following an in vitro kinase assay with cold ATP. Membranes were immuno-blotted with BAK1 antibodies for loading control. c, Trans-autophosphorylation of kinase-dead (D416N) BAK1 substrate (6xHis-BAK1*^CD) at S612 by BAK1-WT (MBP-BAK1^CD). BAK1-Y403 or-S612 phosphorylation was detected using phosphorylation specific antibodies following an in vitro incubation of the purified proteins in the presence or absence of cold ATP. Membranes were immuno-blotted with BAK1 antibodies for loading control. d, [³²P] γ-ATP kinase assay showing autophosphorylation of the indicated affinity purified recombinant BAK1^CD mutants. e, [³²P] γ-ATP kinase assay showing trans auto-phosphorylation of the kinase-dead BAK1-D416N (6xHis-BAK1*^CD) by wild type BAK1 (MBP-BAK1^CD). f, [³²P]γ-ATP kinase assay showing transphosphorylation activity of the indicated affinity purified recombinant BAK1^CD mutants against a kinase-dead (K105E) BIK1 substrate (GST-BIK1*). Numbers indicate autoradiograph band intensity relative to WT. Protein loading control was determined with Coomasie brilliant blue (CBB) staining. a-f, All experiments were repeated independently at least three times. For blot source data, see Supplementary Figure 1.