a, Clustal Omega multiple alignments were visualized using JalView v2.10.2b2. The alignment is coloured by percentage identity. Yellow, conservation of BAK1-S602, -S604 and -S612; green, conservation of BAK1-T603. Protein IDs used for the alignments: PpSERK1(B9MW41), PpSERK2(B9IQM9), PpSERK3(B9HFX1), PpSERK4(B9H599),DlSERK(B5TTV0), MtSERK1(Q8GRK2), MtSERK2(E2IXG1), MtSERK3(E2IXG8), MtSERK4(E2IXG2), MtSERK5(E2IXG3), MtSERK6(E2IXG4), GmSERK1(C6ZGA8), GmSERK2(C6FF61), CuSERK (Q6BE26), CsSERK(C3V9W0), VvSERK1(D7TXV2), VvSERK2(A5BIY4), VvSERK3(D7STF6), CpSERK1(A7L5U3), CpSERK2(E5D6S9), GhSERK1(E5Q8K6), GhSERK2(F5BZU9), GhSERK3(F6MF11), StSERK(A3R789), SpSERK(A6N8J2), SlSERK1(G0XZA3), SlSERK3A(G0XZA5), SlSERK3B(G0XZA6), DcSERK(O23921), AtSERK1(Q94AG2), AtSERK2(Q9XIC7), AtSERK3(Q94F62), AtSERK4(Q9SKG5), AtSERK5(Q8LPS5), NbSERK3A(E3VXE6), NbSERK3B(E3VXE7), OsbiSERK(Q6S7F1), OsSERK(Q5Y8C8), OsSERKlike1(Q67X31), OsSERKlike2(Q6K4T4), SbSERK1(C5YHV3), SbSERK2(C5Y9S6), SbSERK3(C5XVP5), AcSERK1(H6SU43), AcSERK2(H6UP78), AcSERK3(H6UP79), ClSERK(G2XLB1), CnSERK(Q5S1N9), ZmSERK1(Q93W70), ZmSERK2(Q94IJ5), ZmSERK3(B4G007), TaSERK1(G4XGX1), TaSERK2(G4XGX2), TaSERKlike3(G4XGX3), SmSERK1(D8SBB8), SmSERK2(D8S0N3), SmSERK3(D8S4M4), SmSERK4(D8R6C9), MpSERK(A7VM18), PpSERK1(A9STU8), PpSERK2(A9SMW5), PpSERK3(A9RY79), PoapSERKlike1(Q659J0), PoapSERKlike2(Q659J1), CeSERK(A7VM46). b, WebLogo representation of alignment in (a).
c, Detection of BAK1-S612 phosphorylation using α-pS612 specific antibodies on affinity-purified recombinant BAK1CD following an in vitro kinase assay with cold ATP. Membranes were immuno-blotted with α-pS612 and α-MBP antibodies. d, BAK1 immunoprecipitation (IP) and detection of BAK1-S612 phosphorylation in vivo using α-pS612 antibodies. Two-week-old seedlings were treated with water (−) or 100 nM flg22 (+) for 10 min. The same membrane was stripped and blotted again with α-BAK1 antibodies for loading control. e, Western blot analysis using α-BAK1 and α-pS612 antibodies after SDS-PAGE of crude protein extracts from two-week-old seedlings treated with 100 nM flg22 for 10 min. Blots were stained with Coomassie brilliant blue (CBB) for loading control. a-e, All experiments were repeated at least twice with similar results. For blot source data, see Supplementary Figure 1.