Fig. 5. Inhibition of poly(ADP-ribose) synthesis in cells by overexpression of the apoptotic (24 kDa) DBD of PARP-1. (A) Immunoblotting of the overexpressed DBD of PARP-1. Cultured cells were incubated for 16 hours with 1 mM dexamethasone to induce the expression of the DBD, and then extracted in SDS-PAGE loading buffer. 100,000 cells were loaded per well and analyzed by western blotting and immnodetection with the monoclonal antibody (F1-23), which recognizes the second zinc finger of PARP-1. (B) Transdominant inhibition of poly(ADP-ribose) synthesis by the apoptotic DBD of PARP-1 in cells treated with MNNG for 30 minutes. Different concentrations of MNNG were used as indicated on the histogram. The inhibition of pADPr synthesis is expressed as the % fraction of polymers detected in the presence of the 24 kDa DBD (+dexamethasone) over that detected in the absence of the fragment (-dexamethasone). The polymer levels detected in the absence of dexamethasone induction represent 100% PARP activity and are as follows: 185±3.7 units, 0 µM MMNG; 215±8.7 units, 20 µM MMNG; 430±17.3 units, 40 µM MMNG; and 837±15.6 units, 60 µM MMNG. Polymer levels were measured by quantitative immunodot blot (Affar et al., 1998) and are expressed as arbitrary fluorescence units per 4x106 cells. The results plus s.d. of three independent experiments are shown.
