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. 2013 Aug 13;64(14):4343–4360. doi: 10.1093/jxb/ert241

Fig. 1.

Fig. 1.

SUMO–AtACS7 was phosphorylated by AtCDPK16 in vitro. Recombinant protein 6His-SUMO–ACS7 was used as a substrate, and four kinds of recombinant kinases (G-AtCDPK1-6His, G-AtCDPK16-6His, G-AtCRK3-6His, and G-AtCDPK34-6His) were used to perform the kinase assay in vitro. The result indicates that 6His-SUMO–AtACS7 can only be phosphorylated by G-AtCDPK16-6His in vitro. G-NR is a fusion protein, with GST fused with a peptide of nitrate reductase (TLKRTASTPFM), and this peptide is known to be recognized by G-AtCDPK1-6His; G-NRV is a vector-only protein; G-Di19-2-2WT is also a fusion protein in which GST is fused with a peptide (DVLKSEQKEMSYREDPY); this peptide can be recognized by G-AtCDPK16-6His, and G-Di19-2-2MT is similar to G-Di19-2-2WT but with serine mutated to alanine. The molecular weights of all the fusion proteins are ~55kDa and that of SUMO–ACS7 is ~63kDa; the peptide phosphorylation signal is marked with a arrowhead, and the kinase autophosphorylation signal is ~100kDa (right panel).