Fig. 1

Determination of the enzymatic activity of AtPARP3. a Recombinant AtPARP3 activity determination. The purified proteins were incubated with 500 nM broken DNA and 1 mM NAD⁺ at 25 °C for different time periods with or without 20 mM 3-AB. After electrophoresis, immunoblotting was performed with anti-pan-ADPR reagent or other internal control antibodies. The tag proteins, TRXH and GST, expressed by the empty vectors pET32a(+) and pGEX-4 T-1, respectively, were used as the negative controls for the assay. The Coomassie blue-stained SDS-PAGE gel in the bottom panel shows the loading amounts of TRXH (black arrow), GST (white arrow), TRXH-AtPARP1 (red arrow), TRXH-AtPARP3 (green arrow), and GST-AtPARP3 (orange arrow). b Activity determination under extended time periods. The purified proteins were incubated with 500 nM broken DNA and 1 mM NAD⁺ at 25 °C for different time periods. The poly (ADP-ribosyl) ated proteins were separated on an SDS-PAGE gel and detected by anti-pan-ADPR reagent (the upper panel) and anti-AtPARP3 and anti-AtPARP1 antibodies, respectively