Fig. 3.

AtABF3 is phosphorylated by AtCDPKs at its phosphorylation site, in vitro. Recombinant fusion peptides [ABF3 P1 (LQRQGpSLpTLPR), ABF3 P1 MT (LQRQGpSLALPR), ABF3 P3 (LPRTIpSQKRVD), and ABF3 P4 (QCLRRTLpTGPW)] were used as substrates, and two recombinant kinases (AtCDPK16-6His, AtCDPK3-6His) were used to perform the kinase assay. The molecular weight of the full-length recombinant GST-ABF3 protein is about 76kD. The molecular weights of several different fusion proteins are about 55 kD. The fusion peptide phosphorylation signal is marked with an arrowhead, and the kinase auto-phosphorylation signal is about 100 kD. The results showed that that ABF3 could be phosphorylated by a AtCDPK3-6His and b AtCDPK16-6His, in vitro. The ACA2 wild-type (WT) is a fusion peptide in which the GST protein is fused with a peptide from ACA2, RFRFTANLpSKRYEA, which could be recognized by AtCDPK3-6His. The ACA2 mutant (MT) is similar to ACA2 WT, but a Serine was mutated to Alanine. Di19-2-2 is a fusion peptide with a GST protein that is fused with a peptide (DVLKSEQKEMpSYREDPY), and this peptide is recognized by AtCDPK16-6His