Article
Extended Data Fig. 3 | Plasma membrane localization and phosphorylation
are required for BIK1 ubiquitination. a, The kinase inhibitor K252a blocks
f lg22-induced ubiquitination of BIK1. Protoplasts transfected with FLAG–UBQ
and BIK1–HA were treated with 1 μM K252a for 30 min and then with 100 nM
f lg22. b, BIK1(G2A) no longer localizes to the plasma membrane. BIK1–YFP or
BIK1(G2A)–YFP was expressed in N. benthamiana for imaging analysis.
c, BIK1(G2A) show compromised flg22-induced monoubiquitination. BIK1–HA
or BIK1(G2A)–HA was co-expressed with FLAG–UBQ in protoplasts. d, Single
K-to-R mutations of BIK1 fail to block f lg22-induced ubiquitination without
altering kinase activity. HA-tagged wild-type or mutant BIK1 was co-expressed
with FLAG–UBQ in protoplasts. e, BIK1(K204R) exhibits reduced
autophosphorylation and phosphorylation of BAK1. An in vitro kinase assay
was performed using GST–BIK1 or GST–BIK1(K204R) as a kinase and GST or
G S T– B A K 1K (BAK1 kinase domain without detectable autophosphorylation
activity) as a substrate with [γ-^32 P] ATP. Top, proteins were separated with SDS–
PAGE and analysed by autoradiography (Autorad.); bottom, protein loading
shown CBB staining. Experiments were repeated at least twice with similar
results.