Recombinant parp 1

5 pmol of recombinant GST-TET1 or recombinant GST-tag (SignalChem) were incubated with 25 μl of PBS-washed Glutathione Sepharose 4B (GE Healthcare) for 1 hr in rotation at 4°C. After washing with equilibration buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl₂), 5 pmol of unmodified recombinant PARP-1 (Enzo Life Sciences) or automodified recombinant PARP-1 were added to equilibration buffer and incubated with GST-TET1 or GST for 2 hrs in rotation at 4°C. Samples were washed 10 times with wash buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl₂, 150 mM NaCl, 0.1% NP-40, 1% glycerol) and elution was obtained boiling with one volume of 2X Laemmli sample buffer. PARP-1 automodification was obtained incubating 5 pmol of recombinant PARP-1 in PARP activity buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl₂, 0.2 mM DTT, 3 μM NAD⁺) in presence of 200 ng of DNAse I activated DNA (Enzo Life Sciences) for 45 min at 25°C. GST pull-down was also performed in presence of 300 μg of nuclear proteins with an incubation of 4 hrs in rotation at 4°C.

For in vitro ADP-ribosylation, 200 ng of recombinant PARP1 (Enzo Life Science) were suspended in 200 μL reaction buffer (100 mM Tris pH 8, 10 mM MgCl₂, 1 mM DTT). Fourty nM β-NAD and 60 nM ³²P-β-NAD (Perkin Elmer) were added, the reaction was incubated for 30 min at 37°C while shaking, and stopped by Proteinase K digestion at 42°C for 1 h. Resulting poly(ADP-ribose) polymers were subjected to phenol-chloroform-extraction, resuspended in TBS-T (Tris buffered saline - 0.1% TWEEN), and stored at −20°C.
For the dot-blot, a native nitrocellulose membrane was soaked in TBS-T for 10 min and left to air-dry. Two, 4, 8, and 16 pmol of ALC1 and BSA were dot-blotted onto the nitrocellulose membrane and the blot was left to dry for 2 h. Upon blocking for 1 h in TBS-T containing 10% fatty-acid free milk, the membrane was incubated in poly(ADP-ribose) polymer solution at 4°C overnight. The next day the membrane was washed with TBS-T containing 150 μM NaCl for 1 h and exposed to Fuji-X X-ray film for 5 days before imaging with a Fuji-X Bio-Imager.

7 pmol of GST-TET1 (SignalChem) or recombinant GST-tag were incubated with 25 μl of PBS-washed Glutathione Sepharose 4B (GE Healthcare) for 1 hr in rotation at 4°C. After washing with equilibration buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl₂), 3.5 pmol of recombinant PARP-1 (Enzo Life Sciences) were added to PARP activity buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl₂, 0.2 mM DTT, 3 μM NAD⁺) in presence of DNAse I activated DNA (Enzo Life Sciences) and incubated with GST-TET1 or GST at 25°C for different time. Reaction was stopped adding one volume of 2X Laemmli sample buffer. For in vitro TET activity of covalently PARylated GST-TET1, elution was performed in 50 mM Tris-HCl pH 8.0 and 20 mM reduced glutathione after extensive incubations with detergent containing buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl₂, 200 mM NaCl, 0.1% NP-40, 1% glycerol) to wash out PARP-1 protein. Experiments were also performed without DNAse I activated DNA incubating 1 pmol of GST-TET1 or GST in presence of 0.4 pmol of PARP-1 for different time or incubating 9, 1.8, 2.7, 3.6 pmol of GST-TET1 or GST in presence of 9 pmol of PARP-1 for 15 min at 25°C.