Parp1

Mouse cerebellar tissues and cell pellets were homogenized with a cell lysis buffer (Cell Signaling) with Halt phosphatase inhibitor (Thermo-Fisher), complete protease inhibitors (Roche) and PSMF (Sigma-Aldrich). The supernatant was recovered by centrifugation (16,000g, 15mins). Protein concentration was assayed by the Bradford protein assay system (Bio-rad). Twenty micrograms of lysates were loaded into 4–12% Bis—Tris gels (Invitro-gen). Electrophoresis was carried out according to the manufacturer’s recommendations. Following electrophoresis, the proteins were transferred to nitrocellulose membranes by the iBlot device (Invitrogen), blocked with an Odyssey blocking buffer (LI-COR Biotechnology) for 1 h. Membranes were incubated overnight with the following primary antibodies in blocking buffer: Iba1(Wako), CD11b(Abcam), MUTYH(Abcam), PARP-1(Trevigen), Frataxin (Santa Cruz), Actin(Abcam), AT1(Abcam), Tubulin (Abcam). Subsequently, the membranes were incubated with a corresponding pair of IRDye 680CW and IRDye 800CW-coupled secondary antibodies (LI-COR). Proteins were visualized with the Odyssey infrared imager and software (LI-COR) according the manufacturer’s instruction.

In vitro ADP-ribosylation assays were performed as previously described (Gibbs-Seymour et al., 2016 (link)). For autoradiography analyses, recombinant proteins or synthetic peptides were mixed with PARP-1 and/or HPF1 WT or mutants before the addition of activated DNA, 5 μM NAD⁺ and 63nM (0.05 μCi/μl) ³²P-NAD⁺ to the reaction, which proceeded for 20 min at room temperature. Olaparib (2μM final concentration) was added at the end of the reactions before subsequent analysis by autoradiography. The molarity of HPF1 proteins used in the reactions were 1-2 μM, PARP-1 or PARP-2 were 0.1 μM and trans substrates were typically used at 2μM. The recombinant H3 and synthetic peptides substrates were 2μg per condition.
For mass spectrometry analyses, recombinant proteins or recombinant human mononucleosome or synthetic peptides were mixed with PARP-1 (Trevigen) or in-house produced recombinant PARP-1 or PARP-2 from E.coli, and/or HPF1 WT before the addition of activated DNA and 200 μM NAD⁺ to the reaction, which proceeded for 20 min at room temperature. Olaparib (2μM final concentration) was added at the end of the reactions before subsequent trypsin digestion. The molarity of HPF1 WT used in the reactions were 2 μM, PARP-1 and PARP-2 was 0.1 μM. The mass of substrates that we were interested in analyzing their modification sites ranged from 1 to 50 μg per reaction.

10 μg of GST or GST-BAZ2B PHD–BRD was incubated with 5 units of PARP1 (Trevigen, 4668-500-01) in 250 μl of pulldown buffer (20 mm Tris (pH 7.5), 100 mm NaCl, 0.05% Triton, and protease inhibitors) for 2 h with rotation at 4 °C. 20 μl of glutathione-agarose (Thermo Fisher Scientific) was added and incubated for an additional 2 h. The beads were washed four times with 300 μl of pulldown buffer and eluted by boiling in SDS sample buffer.