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. 2016 Aug 21;16(6):fow068. doi: 10.1093/femsyr/fow068

Figure 6.

Figure 6.

Phosphorylation of CnYpd1, CnYpd1 E58A and CnYpd1 D60A-E67A. Phospho∼ScSln1-HKR1 was used as a donor to CnYpd1 constructs and removed from the reaction after 2 min. (A) Autophosphorylation of bead-bound ScSln1-HKR1 was performed in the presence of 10 mM MgCl2. Bead-bound ScSln1-HKR1 was split into two fractions and each was washed by gentle pelleting of resin and addition of reaction buffer with either 10 mM MgCl2 or CaCl2. CnYpd1 was added to each ScSln1-HKR1 in the presence of CaCl2(A) or MgCl2(B) containing tube, ScSln1-HKR1 was removed from the reaction after 1 min and aliquots were taken of only CnYpd1 at 1, 5 and 15 min (lane designations indicate time points). (C) CnYpd1 E58A and CnYpd1 D60A-E67A were phosphorylated using ScSln1-HKR1. ScSln1-HKR1 was removed from the reaction after 1 min and aliquots were taken. Lane 1: WT CnYpd1, Lane 2: CnYpd1 E58A and lane 3: CnYpd1 D60A- E67A.