Brassinosteroids (BRs) are plant steroid hormones sensed at the cell surface by the membrane receptor kinase BRI1. Activation of BRI1 leads to the dephosphorylation of BZR1/BES1 transcription factors through inactivation of the glycogen synthase kinase 3 BIN2. Here, the in vitro phosphorylation sites of the isolated BSU1 Kelch domain (residues 11-351), phosphatase domain (residues 421-793) and of full-length BSU1 (residues 1-793) recombinantly expressed in baculovirus-infected Spodoptera frugiperda cells were identified in isolation (labelled ctrl) or after incubation with the GSK3 kinase BIN2, expressed as a thioredoxin A fusion protein in E. coli. Reactions were performed in the presence of 100 micromolar ATP and 1 mM MgCl2 for 1:30 h at room temperature.