The JNK1 kinase interacts with NEIL1.

<p>(A) Western blot analysis of endogenous JNK1 present in HEK293T WCE and in NEIL1-FLAG immunoprecipitates. (B) Far-Western analysis to confirm the interaction between NEIL1 and JNK1. Top panel, Coomassie stained SDS-PAGE gel of NEIL1 and its phosphomimetic and ablating mutants (50 pmoles). Bottom panel, membrane after transfer, denaturation, slow refolding, binding to JNK1 from HEK293 WCE, and probing with an anti-JNK1 antibody. BSA was used as a negative control.</p

Binding of NEIL1 and mutant enzymes to DNA.

<p>(A) Representative phosphor-autodiogram for binding of NEIL1-WT to a 35-mer furan-containing DNA where the substrate (10 pM) was incubated with increasing (0–600 nM) amounts of enzyme. Complex formation is indicated by the presence of shifted bands C1 and C2. (B) Graphical fitting of the EMSA data indicated above using GraphPad Prism 6. The data were fit to the one-site specific binding equation. The K_d values for WT and the NEIL1 mutants are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157860#pone.0157860.t001" target="_blank">Table 1</a> and are representative of experiments performed in duplicate.</p

Phosphorylation of NEIL1 by JNK1 using an <i>in vitro</i> kinase assay.

<p>(A) <i>In vitro</i> kinase assays were performed with NEIL1 constructs expressed from E. coli cells and purified to homogeneity and active JNK1 kinase for 30 minutes at 32°C. γ-³²P incorporation was quantified using phosphor-autoradiography after SDS-PAGE analysis of the samples. Lane 1, no JNK1 control; lane 2, no NEIL1 control; lane 3–13 are WT, S207E, S207A, S306E, S306A, S61E, S61A, DE, DA, TE, TA, respectively. (B) Graphical representation of two experimental repeats of the <i>in vitro</i> kinase assay. Statistically significant values (at 95% confidence) were determined by a one-way Anova test where * denotes p-values <0.05, *** denotes p-values <0.0005, and **** denotes p values <0.0001. (C) Glycosylase activity assays were performed using Sp:C and AP:C substrates with increasing amounts of <i>in vitro</i> phosphorylated NEIL1 (pNEIL1), unphosphorylated NEIL1 (positive control), and JNK1 (negative control). S and P indicate substrate and product, respectively.</p

Sites of phosphorylation within the NEIL1 DNA glycosylase.

<p>(A) Domain map of NEIL1 indicating the position of known sites of phosphorylation. The residues S207, S306, and S61 identified in this study are shown in blue and the Y263 and S269 sites previously identified [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157860#pone.0157860.ref039" target="_blank">39</a>] are indicated in black. (B) SDS-PAGE gel of SBP-tagged NEIL1 after affinity pull-down from HEK293T cell-extracts overexpressing NEIL1. The gel was stained with Coomassie blue and the NEIL1-SBP band was cut from the gel and digested with trypsin for identification of phosphorylated peptides via LC-MS/MS.</p

Phosphomimetic/ phosphoablating mutants of NEIL1.

<p>Phosphomimetic/ phosphoablating mutants of NEIL1.</p

Identification of phosphorylation sites on NEIL1 by LC-MS/MS.

<p>(A) List of phosphorylated peptides identified from LC-MS/MS analysis of NEIL1-SBP with their corresponding monoisotopic mass m/z, charge-state (z), and Xcorr values. (B) Product-ion spectra (Scaffold software 4.3) for each phosphorylated peptide are displayed. The b- and y-ions are shown in red and blue, respectively, and the neutral loss peak resulting from the loss of a phosphogroup from the parent ion is indicated in green.</p

Glycosylase and lyase activity panel for human NEIL1-WT and the phosphomimetic/ablating mutants.

<p>Glycosylase assays were performed by incubating 20 nM of double-stranded DNA substrates (A) Sp:C and (B) AP:C and increasing amounts of enzyme with the following substrate to enzyme ratios: 1:0.5, 1:1, 1:4, and 1:16. “-” indicates a no enzyme negative control. Assays were performed at room temperature for 30 minutes. S and P indicate substrate and product, respectively. Data shown are representative of duplicate experiments.</p