PARP1 HSA (trevigen) was incubated in the presence or absence of A3, ABT888, or equal volume of DMSO at indicated concentrations in PARylation buffer (20 mM Tris 7.9, 100 mM NaCl, 10 mM MgCl2, 10 mM ZnCl2, 10% Glycerol, 1 mM DTT, 1 mg/ml ssDNA (Sigma), 300 uM B-NAD (Amresco)) for 30′ at 30 degrees C. Negative control reactions are performed in the absence of B-NAD. 2x SDS PAGE sample buffer stops the reaction and sample is loaded on SDS-PAGE for subsequent western-blotting for PAR and PARP1.
Brodie S.A., Li G., Harvey D., Khuri F.R., Vertino P.M, & Brandes J.C. (2015). Small molecule inhibition of the CHFR-PARP1 interaction as novel approach to overcome intrinsic taxane resistance in cancer. Oncotarget, 6(31), 30773-30786.
+ Open protocol