Enzymatic Activity Assay for L230

L230 enzymatic activity was measured as recently described^{22 (link)}. In brief, the assay was performed in reaction buffer (50 mM HEPES buffer pH 7.4, 150 mM NaCl) at 37 °C for 1 h with 1 μM L230 enzyme, 10 μM FeSO4, 100 μM 2-OG, 500 μM ascorbate, 1 mM dithiothreitol, 0.01% Triton X-100, and 1 mM viral collagen peptide substrate ETGLKGII or 4 μM bovine skin collagen substrate containing no telopeptides (Bovine PureCol^®, Advanced BioMatrix). With the exception of L230 recombinant protein and bovine skin collagen, all reagents were prepared immediately before use. All of these reagents were dissolved in reaction buffer with the exception of FeSO₄ and collagen, which was prepared in 10 mM HCl, and the pH of the reaction mixture was checked with pH papers to ensure that HCl did not change the overall sample pH. Bovine skin collagen was denatured by heating at 95 °C for 5 min and then chilled immediately on ice before use. L230 activity was measured by detecting succinate production with an adenosine triphosphate-based luciferase assay (Succinate-Glo™ JmjC Demethylase/Hydroxylase Assay, Promega, Madison, WI) according to the manufacturers’ instructions. Results shown are the mean values of triplicate biological samples in a single experiment. Each experiment was repeated once.