Research Paper Volume 11, Issue 9 pp 2852—2873

Figure 4. Influence of exogenous proteins on (ADP-ribosyl)ation of primer in WCEs. (A) WCE proteins in the absence or presence of extra recombinant proteins were incubated for 5 min with 100 nM DNA duplex dRP, 0.5 mM NAD⁺, and 5 mM spermine as described in the section ‘DNA (ADP-ribosyl)ation assay’. Recombinant PARP1, PARP2, or PARP3 were added to the extracts at the indicated concentrations prior to initiation of the (ADP-ribosyl)ation reaction. Lane 1, no extra recombinant proteins were added; lane 12, no extract proteins were added. (B) Mmu or HEK293T WCE proteins (0.5 mg/mL) were incubated for 5 min with 100 nM DNA duplex containing dRP moiety in the presence of 0.5 mM NAD⁺ and 5 mM spermine as described in the section ‘DNA (ADP-ribosyl)ation assay’. PARP3 and/or Ku (each at the final concentration of 300 nM) were added to the extracts prior to initiation of the (ADP-ribosyl)ation reaction. Lane 1 corresponds to the initial primer (control). (C) Mmu or HEK293T WCE proteins (0.5 mg/mL) were incubated at 37 °C for 5 min with 100 nM DNA duplex containing the dRP moiety in the presence of 0.5 mM NAD⁺ as described in the section ‘DNA (ADP-ribosyl)ation assay’. PARP1, PARP1^E988K, PARP2, or PARP3 (the final concentrations of 300 nM) were initially added to the reaction mixtures and indicated. 50 nM PARG was added to some mixtures (lanes 6 and 8) after the reactions were stopped by the addition of EDTA, and the mixtures were incubated at 37 °C for another 10 min.