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. 2020 Jun 5;183(4):1825–1837. doi: 10.1104/pp.20.00184

Figure 4.

Figure 4.

Fls3 and Fls2 vary in their kinase activity in vitro. The KD alone or the iJM plus the KD (JMKD) of Fls3 and Fls2 were expressed as recombinant proteins in the pDEST-HisMBP vector and tested for in vitro kinase activity. The kinase-inactive mutants for Fls3(K877Q) and Fls2(K900Q) were generated in the background of the JMKD constructs. HisMBP protein was used as a negative control. A, Fls3 or Fls2 KDs or JMKDs were tested in in vitro kinase assays. B, MyBP was added to the in vitro kinase assays to test the ability of the kinases to transphosphorylate the MyBP substrate. C, Kinase assays swapping the iJMs of Fls3 and Fls2 to test the requirement for their cognate iJM. 3JM-2KD expresses the iJM from Fls3 and the KD from Fls2, and 2JM-3KD expresses the iJM from Fls2 and the KD from Fls3. Data shown are representative of at least three independent experiments. Exposure length to the phosphor-screen (4 or 14 h) and the SDS-PAGE gel percentage are indicated.