Fuji x bio imager

Newly prepared GST-vector or GST-Smad proteins were kept on glutathione beads and incubated in 100 µl PARP-1 reaction buffer (100 mM Tris-HCl [pH 8], 10 mM MgCl₂, 1 mM dithiothreitol), with or without 100 ng PARP-1 or 100 ng PARP-2. Then, 80 nM β-NAD and 20 nM ³²P-β-NAD were added and the samples were incubated for 30 min at 37°C while shaking. For reactions with excess cold NAD, instead of 80 nM β-NAD, 180, 480 or 980 nM β-NAD were included in separate reactions, reaching the total concentration of cold plus radioactive β-NAD to 200, 500 and 1,000 nM respectively (Fig. S1). PARG incubations were performed in PARG reaction buffer containing (100 mM Tris-HCl, pH 8.0, 10 mM MgCl₂ and 1 mM dithiothreitol) with and without PARG. At the end of each reaction, beads with GST fusion proteins were collected via centrifugation, followed by a quick double wash in ice-cold NP-40 lysis buffer to remove excess radioactive β-NAD. Samples were then heated for 4 min at 95°C in sample buffer and subjected to SDS-PAGE. Gels were fixed, stained with CBB and dried before measuring radioactivity in a Fuji-X Bio-Imager (FujiFilm Corp., Stockholm, Sweden).

Newly prepared GST or GST-Smad proteins were kept bound to glutathione beads and incubated in 100 μl of reaction buffer (100 mm Tris-HCl, pH 8, 10 mm MgCl₂, 1 mm DTT), with or without 100 ng of PARP1 (Axxora, LLC/ENZO Life Sciences). Then 80 nm β-NAD⁺ and 20 nm³²P-β-NAD⁺ (PerkinElmer) were added, and the reactions were incubated for 30 min at 37 °C while shaking. At the end of each reaction, beads bound to GST fusion proteins were collected via centrifugation, followed by a quick double wash with ice-cold Nonidet P-40 lysis buffer to remove excess radioactive β-NAD⁺. Alternatively, a sample of the total reaction was collected. Samples were then heated for 4 min at 95 °C in sample buffer and loaded on gels. Gels were fixed, stained with Coomassie Brilliant Blue, and dried before measuring radioactivity in a Fuji-X Bio-Imager. The non-radioactive version of the same assay was performed exactly in the same manner, except that a total of 100 nm cold β-NAD⁺ was included in the reaction.

Freshly prepared GST and GST-Smads were immobilized on glutathione beads with 100 μl of reaction buffer (100 mm Tris-HCl, pH 8, 10 mm MgCl₂, 1 mm DTT) along with 100 ng of PARP1 and increasing amounts of PARG (Axxora, LLC/ENZO Life Sciences). Then 80 nm β-NAD⁺ and 20 nm³²P-β-NAD⁺ were added to the mixture, and samples were incubated at 37 °C while shaking. After 30 min, beads were centrifuged at 4,000 rpm and washed three times with ice-cold Nonidet P-40 lysis buffer to remove unbound PARG, PARP1, and β-NAD⁺. Washed beads were then mixed with sample buffer with β-mercaptoethanol and heated for 4 min at 95 °C and loaded on gels. Gels were then fixed, stained with Coomassie Brilliant Blue, and dried to measure the radioactivity by a Fuji-X Bio-Imager.