Research Paper Volume 4, Issue 10 pp 674—685

Figure 1. NEIL1 binds to PARP-1.

(A) HeLa cells were lysed and incubated with GST, GST-OGG1 or GST-NEIL1 (20 μg) and then were either treated with DNase I (+) or mock treated (−). Samples were analyzed on a polyacrylamide gel, stained with Ponceau S and probed with anti-PARP-1 antibodies. ~1.5% of total PARP-1 bound to GST-NEIL1 and ~4% of total PARP-1 bound to GST-OGG1. (B) GST, GST-OGG1 and GST-NEIL1 (1 μg) were incubated with 250 ng purified PARP-1 in vitro. Samples were treated with DNase I and immunoblotted with anti-PARP-1 and anti-GST antibodies. ~5% of purified PARP-1 bound to both GST-OGG1 and GST-NEIL1. (C) HeLa cells were transfected with pEGFP or pEGFP-NEIL1 and mock treated (−) or treated (+) with 500 μM H₂O₂. PARP-1 immunoprecipitates were probed with anti-PARP-1 and anti-GFP antibodies. Arrows indicate GFP-NEIL1, immunoglobulin heavy chain (IgGH) and immunoglobulin light chain (IgGL) in the immunoprecipitates and GFP and GFP-NEIL1 in the lysates. ~1.5% of total PARP-1 was immuno-precipitated with GFP-NEIL1. There was no significant change in GFP-NEIL1 binding to PARP-1 after H₂O₂ treatment (P=0.25, n=3). Representative immunoblots are shown for each experiment and repeated in at least 3 independent experiments.