Fig. 1

Loss-of-PARP2 affects the branched PAR chain formation. a Diagram depicting the procedure of sample preparation for dot blot, UV spectroscopy or mass spectrometry. Wide type or Parp2^−/− MEFs were treated with 500 μM H₂O₂ for 10 min to induce DNA damage. PAR was extracted and followed by digestion with pyrophosphatase (PPase) and alkaline phosphatase (AP) prior to LC–MS/MS. b Dot blot assays were performed with anti-PAR antibody. 5X means five-fold loading samples. Actin was used as the control of cell lysates (monoclonal antibody, Sigma A2228). c Depletion of PARP2-induced minor reduction of total PAR in response to DNA damage, whereas lacking PARP1 remarkably suppressed total PAR level. The levels of PAR were examined at 259 nm using UV spectroscopy. d Mass spectrometry detection of adenosine (Ado), ribosyladenosine (R-Ado) and diribosyladenosine (R₂-Ado). LC–MS/MS quantitative analysis showed that Ado (terminal PAR unit) and R₂-Ado (branched PAR unit) were remarkably reduced in PARP2-deficient cells, but not R-Ado (linear PAR unit). e Only full length PARP2, but not the E545A mutant restores the branched PAR chain formation. PARP2 was deleted in U2OS cells and reconstituted with either full length PARP2 (ΔPARP2 + FL) or the E545A mutant (ΔPARP2 + E545A, left panel). The levels of R₂-Ado were measured by LC–MS/MS (middle and right panels). Data are represented as mean ± s.d. as indicated from three independent experiments. Significance of differences was evaluated by Student’s t-test. NS: non significant; *statistically significant (p < 0.05); ***statistically significant (p < 0.001)