2.8. PARP activity assay

The PARP activity assay was performed using the Trevigen PARP activity kit. All reagents were supplied by the kit unless otherwise specified. The procedures were previously described by Sun et al., 2012. Basically, cells were lysed with the Cell Extraction Buffer supplied in the kit, and the protein concentration was determined by the BCA Protein Assay. Two hundreds ng of total proteins from each sample was combined with activated DNA and nicotinamide adenine dinucleotide (NAD) supplied by the kit, and then placed into a histone-coated strip well to formed PAR complex which was fixed to the bottom of the well. Anti-PAR monoclonal antibody was then added to the well to bind to PAR complex, followed by a Horseradish peroxidase (HRP) conjugated secondary antibody against the primary antibody. TACS- Sapphire™ was used a substrate to generate the chemiluminescence signal. An equal amount of 0.2 M HCl was added to stop the reaction. The signals were detected using SpectraMax® 340PC microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm wavelength.