Research Paper Volume 4, Issue 10 pp 674—685

Figure 4. NEIL1 stimulates the poly(ADP-ribosyl)ation activity of PARP-1.

(A) PARP-1 activity was measured by determining the amount of PAR deposited on immobilized histones in an ELISA assay. Addition of NEIL1 (33 ng) increased the amount of PAR synthesis by PARP-1 (2 ng). The histogram represents the mean + SEM from three independent experiments. *p < 0.05 compared to control by Student's t-test. (B,C) GST, GST-NEIL1, GST-NEIL1 1–288 aa or GST-NEIL1 289–390 aa (5 μg) were incubated with (+) or without (−) 7.5 ng PARP-1. The immunoblot was stained with Ponceau S and probed with anti-PAR antibodies. (D) WT or NEIL1−/− MEFs were mock treated (−) or treated (+) with 500 μM H₂O₂. Lysates were probed with anti-PAR antibodies and reprobed with anti-PARP-1 and anti-GAPDH antibodies as a protein loading control. (E) NEIL1−/− MEFs transfected with pEGFP-NEIL1 or pEGFP control were mock treated (−) or treated (+) with 500 μM H₂O₂ and lysates were probed as above. Anti-GFP antibodies were used to examine levels of the transfected proteins. For (B-E), a representative experiment from at least 3 independent experiments is shown.