Amp glo assay kit

The ADP-ribosylated peptides were chemically synthesized (table S1). Serine-ADPr and arginine-ADPr peptides were synthesized as previously described (71 (link), 72 (link)). Chemical synthesis of the glutamate-ADPr peptide was previously described (73 (link)). The hydrolytic assay against the ADP-ribosylated peptides was performed as previously described (74 (link)). Briefly, 10 μM substrate peptides (arginine-ADPr, serine-ADPr, or glutamate-ADPr) was hydrolyzed using 1 μM PARP14 MD1, PARP14 MD1mut, PARP14 MD2, PARP14 MD3, PARP9 MD1, PARP9 MD2, or S2 Mac1. ARH1, ARH3, and MacroD1 served as positive controls for arginine-ADPr, serine-ADPr, and glutamate-ADPr, respectively. Hydrolysis was carried out for 1 hour at 30°C in assay buffer [50 mM tris-HCl (pH 7.5), 200 mM NaCl, 10 mM MgCl₂, 1 mM dithiothreitol (DTT), and 0.2 μM Nudix hydrolase 5 (NUDT5) for arginine-ADPr and serine-ADPr and 50 mM Pipes (pH 6.9), 200 mM NaCl, 10 mM MgCl₂, 1 mM DTT, and 0.2 μM NUDT5 for glutamate-ADPr]. The reactions were analyzed using the AMP-Glo assay kit (Promega) following the manufacturer’s recommendations. Luminescence was read using a SpectraMax M5 plate reader with the SoftMax Pro software (Molecular Devices). Data were analyzed using GraphPad Prism.

To quantify the AMP and ATP content in cells, we lysed 1 × 10⁷ pelleted cells by freeze-thaw in liquid nitrogen and subsequent shift to − 80 °C for overnight. Lysed cell pellets were re-suspended in 50 μl of water, centrifuged at 10,000 rpm for 10 min at 4 °C, and supernatants assayed. AMP levels were measured by luminescence detection using the AMP-Glo assay kit (Promega # V5011), following the manufacturer’s instructions. Ten microliters of supernatant was mixed with 10.0 μl of AMP-glow reagent-I for 1 min and incubated at 22 °C for 1 h. Twenty microliters of AMP detection solution was added, mixed, incubated at 22 °C for 1 h, and luminescence measured as described. ATP levels were measured using the ATPlite luminescence assay system (PerkinElmer # 6016943), following the manufacturer’s instructions. Ten microliters of supernatant was mixed with 10.0 μl of ATPlite substrate solution in the dark for 10 min and luminescence measured. We normalized the AMP and ATP levels with protein concentrations in each sample. Two microliters of supernatant was mixed with 100 μl of Bradford protein assay reagent (Bio-Rad # 5000201), incubated for 15 min, and concentration measured by absorbance at 595 nm.