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. 2023 Mar 31;2(3):100090. doi: 10.1016/j.cellin.2023.100090

Fig. 5.

Fig. 5

Enzymatic assays of tandem “HxH” repeats substrate by METTL9.(A) Impact of zinc coordination on METTL9 activity. Unmethylated “HxH”-rich SLC39A755-70 peptide was used as substrate and mono-methylated product was used to monitor the enzymatic activity. Error bars, N = 3, One-way ANOVA with post-hoc analysis. (B) Time-course Q-TOF-MS based enzymatic assays of METTL9 with unmethylated SLC39A755-70 peptide as substrate. Different methylation states are shown as indicated. The concentration of METTL9 is 0.5 μM, and concentration of substrate peptide is 100 μM. (C) ITC fitting curves of SLC39A755-70 peptide with ZnCl2. KD values are listed with standard deviations. ITC were carried out with three biological replicates. (D) Qualitative detection of methylation patterns of remaining tetra-methylated products after 480 min of reaction. Methylation states and modification sites are highlighted in red with underlines. “C-to-N” consecutive methylation patterns are illustrated in right column with red arrows. Due to technical problems, we were unable to detect the LC-MS/MS data of the penta-methylated product. (For Interpretation of the references to colour in this figure legend the readers is referred to the web version of this article.)