3 aminobenzamide 3ab

Human liver cancer cell lines, HepG2 cells (Riken, Tsukuba, Japan) and Li7 cells, were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml) in a humidified incubator with 5% CO₂ at 37.0 °C. The other human liver cancer cell lines, HLE cells, HuH6 cells, HuH7 cells and PLC/PRF/5 cells, were cultured in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml) in a humidified incubator with 5% CO₂ at 37.0 °C. 3-Aminobenzamide (3AB) was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). [³²P]NAD⁺ was purchased from PerkinElmer (Massachusetts, USA), olaparib from ChemScene LLC (New Jersey, USA), recombinant human ADH1 protein (ab123147) from Abcam (Cambridge, UK), and recombinant human PARP1 from Trevigen (Maryland, USA). Poly(ADP-ribose) was prepared as described [16 (link)]. Anti-poly(ADP-ribose) IgG column was prepared by binding of monoclonal antibody against poly(ADP-ribose) (10H) [17 (link)] to CNBr-activated Sepharose 4B (GE Healthcare).

HeLa cells were provided by Riken (Tsukuba, Japan) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml), in a humidified incubator with 5% CO₂ at 37 °C. 3-Aminobenzamide (3AB) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Olaparib was purchased from ChemScene (NJ, USA), emetine hydrochloride from Cayman Chemical (No. 21048; MI, USA), and PDD00017273 [37 (link)] from Tocris Bioscience (MN, USA), and PD0325901 from ChemScene. The following antibodies were obtained from Santa Cruz Biotechnology (TX, USA): mouse anti-human PARP1 IgG (sc-8007), mouse anti-human phosphorylated ERK IgG (sc-7383), mouse anti-human ERK2 IgG (sc-1647), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (sc-2005), and HRP-conjugated goat anti-rabbit IgG (sc-2004). Rabbit anti-human PARG IgG (ab16060) was purchased from Abcam (Cambridge, UK). Anti-PAR (10H, IgG3 kappa), secreted from hybridoma cells [38 (link)], was purified using a Protein A Sepharose Fast Flow column (GE Healthcare, IL, USA). Anti-PAR polyclonal antibodies were produced in rabbits by injecting PAR mixed with methylated BSA in our laboratory [39 (link),40 ]; the antibodies were purified with a Protein A Sepharose Fast Flow column.