E8200

The SINA2 open reading frame (663 bp) was cloned into the pET-59-DEST vector via the Gateway system (Invitrogen) to generate pET-SINA2 containing a 6 × His-SINA2 fusion construct. The PCR product of CDKG1 was inserted into XbaI and PstI sites in a pMAL-c2x vector (New England Biolabs) as pMAL-CDKG1 to produce the N-terminal MBP fusion protein. These recombinant vectors were expressed in E. coli strain BL21 (DE3) by induction overnight with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16 °C. Recombinant 6 × His-SINA2 fusion protein was purified by using Ni agarose beads (GE Healthcare), and MBP-CDKG1 fusion protein was purified according to the pMAL protein fusion and purification system (E8200, NEB). Approximately 5 μg of MBP-AtCDKG1 fusion protein was immobilized on amylose beads and incubated with His-tagged SINA2 proteins. After incubation at 4 °C on a rotary incubator for 2 h, the beads were washed five times with ice-cold buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM DTT). The MBP protein was used as a control in the assay. Bound proteins were re-suspended by boiling in SDS gel-loading buffer for 5 min and separated by SDS-PAGE. After running the gel, protein bound to MBP-AtCDKG1 was detected by Western blotting with anti-His antibody.

IPD3 was expressed from pET28a in E. coli strain Rosetta (TransGen Biotech). Expression products were affinity purified via nickel-agarose (Qiagen) under the denaturing conditions by using 8 M urea. Denatured proteins were refolded by stepwise dialysis26 (link). Purification of MBP-CCaMK, HIS-IPD3, HIS-MtDELLA1, HIS-MtDELLA2 and HIS-MtDELLA3 was performed according to the protocol of E8200 (New England Biolabs) and of BioSprint96 (Qiagen), respectively. IPD3 was phosphorylated in vitro by CCaMK as described26 (link). Each reaction was carried out by using 1 μg MBP-CCaMK protein, 0.2 mM CaCl₂, 0.5 μM Bovine CaM, 2 μg full length and 6 × HIS-IPD3 as substrate. Phosphorylation reactions were performed at 30 °C for 30 min in the presence of DELLA1, DELLA2 and DELLA3. Relative intensities were then calculated for each band by ImageJ.

IPD3 was expressed from pET28a in E. coli strain Rosetta (TransGen Biotech). Expression products were affinity purified via nickel-agarose (Qiagen) under denaturing conditions using 8 M urea. Denatured proteins were refolded by stepwise dialysis (Yano et al., 2008 (link)). Purification of MBP-DMI3, HIS-IPD3, and HIS-IPD3L was performed according to the protocols of E8200 (New England Biolab) and of BioSprint96 (Qiagen). IPD3 was phosphorylated in vitro by DMI3 as described (Jin et al., 2016 (link)). Each reaction was carried out with 1 mg MBP-DMI3 protein, 0.2 mM CaCl₂, 0.5 mM bovine CaM, 2 mg 6XHIS-IPD3 as substrate. Mass spectrometric analysis, in-gel digestion of phosphorylated IPD3, liquid chromatography (LC)-MS data acquisition and database searches were performed by Shanghai Applied Protein Technology Co. Ltd (http://www.aptbiotech.com).