Recombinant proteins were prepared according to (Choi et al., 2017 (link); Lee et al., 2015 (link)), using a modified pMAL vector (New England Biolabs, USA) with maltose binding protein (MBP) and 6X His double tags. Coding regions of relevant proteins were amplified using primers enlisted in Supplementary Table S1 and cloned into the vector. Kinase assays were conducted according to (Choi et al., 2017 (link); Lee et al., 2015 (link)). Site-directed mutagenesis was conducted using the Q5®Site-Directed Mutagenesis Kit (New England Biolabs) according to the supplier’s instruction. Primers are listed in Supplementary Table S1 .
Unless stated otherwise, 0.5 to 1.0 μg of recombinant proteins were used as kinases or substrates. Reactions were conducted at 30°C in a buffer (25 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 10 μM ATP) containing 2 μCi of [γ-32P]-ATP. After the reaction, the mixtures were separated by SDS-PAGE on 10% to 12% gels, and gels were stained with Coomassie Brilliant Blue R (CBB), dried and autoradiographed.
Unless stated otherwise, 0.5 to 1.0 μg of recombinant proteins were used as kinases or substrates. Reactions were conducted at 30°C in a buffer (25 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 10 μM ATP) containing 2 μCi of [γ-32P]-ATP. After the reaction, the mixtures were separated by SDS-PAGE on 10% to 12% gels, and gels were stained with Coomassie Brilliant Blue R (CBB), dried and autoradiographed.
