Pyrophosphate assay kit

Biochemical activity of SUMO specific E1 enzymes was examined by ATPase assay and in vitro gel-shifting assays. ATP hydrolysis activity of E1 enzymes was measured by using pyrophosphate assay kit (Abcam Inc., MA, USA). 0.05 µM E1, 0.25 µM E2, 12.5 µM SUMO-1, and 1 mM ATP with N106 or DMSO were used for the assays. The E1-thioester formation was analyzed in reaction mixtures containing 0.05 µM E1, 12.5 µM SUMO-1, and 1 mM ATP with N106 or DMSO. The E1 dependent E2-SUMO-1 thioester formation was assessed using reaction containing 0.05 µM E1, 0.25 µM E2, 12.5 µM SUMO-1, and 1 mM ATP with N106 or DMSO. Assays were incubated at 37°C for 30 minute and were mixed with non-reducing sample buffer and analyzed in non-reducing condition. Western blot using antibodies against SAE2 and Ubc9 detected the SUMO conjugations. To determine the effect of N106 on Ubiquitin-activating enzyme E1 activity, similar assays were performed (Supplementary Fig.6). Inorganic pyrophosphate detection assays, Ubiquitin-E1 conjugation, and Ubiquitin-E2 conjugation were performed as previously described^{32 (link)}. Human recombinant proteins (wild type SAE1/SAE2, Ubc9, SUMO-1, Ubiquitin, UbcH5A) and reaction buffers were purchased from R&D systems. N106 stock solution for in vitro assays was prepared in DMSO (Sigma-Aldrich, MO, USA).