Anti par monoclonal antibody

For PARP1 inhibition assay, 0.2 μg of recombinant PARP1 was incubated with DMSO (control) or COH34 in a 10-μl reaction mixture [10 mM tris-HCl (pH 8.0), 10 mM MgCl₂, 125 μM NAD⁺, 10 mM dithiothreitol, and octameric oligonucleotide GGAATTCC (5 ng/μl)] for 30 min at room temperature. Samples were analyzed by dot blotting with anti-PAR monoclonal antibody (Trevigen). For TARG1 inhibition assay, mono–ADP-ribosylated PARP10 was generated in a 10-μl incubation mixture comprising 50 ng of GST-tagged PARP10, 100 μM tris-HCl (pH 8.0), 100 μM MgCl₂, 320 μM NAD⁺, and 100 μM dithiothreitol for 30 min at 37°C. Following incubation, GST beads were added to conjugate mono–ADP-ribosylated PARP10 for 2 hours at 4°C and then washed with Mg²⁺-contained tris-HCl buffer (pH 8.0) three times to remove residual NAD⁺. Beads (10 μl) were incubated with 0.5 μM TARG1 and DMSO (control) or COH34 in a 15-μl reaction for 30 min at 37°C. The samples were boiled in the SDS sample buffer at 95°C, and eluates were analyzed by Western blot with anti-adenosine diphosphate ribose antibody (Millipore).

Female NSG mice (8 weeks old; The Jackson Laboratory) were inoculated with 8 million HCC1395 or 10 million PEO-1 cells subcutaneously. Mice were separated into treatment groups (n = 30 per group) of roughly equal tumor size (100 mm³) and dosed with COH34 (20 mg/kg) in 30% solutol or vehicle (30% solutol) once via intraperitoneal injection. Tumor size was measured by an electronic caliper using the standard formula: 0.5 × length × width². After dosing at 0.5, 2, 8, 24, 48, and 72 hours, mice were euthanized using a CO₂ gas chamber, and tumors (n = 5 per time point) were harvested and frozen by dry ice. Tumors were suspended in 500 μl of 1% SDS lysis buffer [1% SDS, 10 mM Hepes (pH 8.5), 500 U of Benzonase (Millipore), and 2 mM MgCl₂] and then homogenized by a Tissuemizer. To remove SDS, sample solution was centrifuged at 1000g at 4°C for 10 min after adding 25 μl of SDS precipitation reagents (Thermo Fisher Scientific). The samples were then analyzed by dot blotting with anti-PAR monoclonal antibody (Trevigen).

The KillerRed system was a gift from L. Lan (University of Pittsburgh School of Medicine, PA). Activation of KillerRed followed the method as previously described (62 (link)). Briefly, U2OS tet response element (TRE) cells with transfection of pBROAD3/tetR-KR were pretreated without or with 0.1 μM COH34 for 1 hour before exposure to a 15-W white fluorescent bulb for 10 min (height to light is 15 cm). Following 1 or 10 min of recovery, samples were examined by immunofluorescence with anti-KillerRed antibody (Evrogen), anti-PAR monoclonal antibody (Trevigen), or anti-XRCC1 antibody (GeneTex). Images were taken from a fluorescence microscope and analyzed by ImageJ.