Journal: PLoS ONE

Article Title: Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase

doi: 10.1371/journal.pone.0157860

Figure Lengend Snippet: (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.

Article Snippet: An anti-mouse JNK1 purified monoclonal antibody (for far-Western analysis, clone 228601 from R&D Systems, lot # UQL0209081; cat # MAB17761) and a rabbit polyclonal NEIL1 antibody (for Western Blotting of IP reactions, Abcam, ab21337, lot # GR133490-1) were diluted 1:1000 (final concentration 1μg/ml) in blocking reagent.

Techniques: Western Blot, Staining, SDS Page, Membrane, Binding Assay, Negative Control

Journal: PLoS ONE

Article Title: Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase

doi: 10.1371/journal.pone.0157860

Figure Lengend Snippet: (A) In vitro 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- 32 P 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 in vitro 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 in vitro phosphorylated NEIL1 (pNEIL1), unphosphorylated NEIL1 (positive control), and JNK1 (negative control). S and P indicate substrate and product, respectively.

Article Snippet: An anti-mouse JNK1 purified monoclonal antibody (for far-Western analysis, clone 228601 from R&D Systems, lot # UQL0209081; cat # MAB17761) and a rabbit polyclonal NEIL1 antibody (for Western Blotting of IP reactions, Abcam, ab21337, lot # GR133490-1) were diluted 1:1000 (final concentration 1μg/ml) in blocking reagent.

Techniques: In Vitro, Construct, Purification, Autoradiography, SDS Page, Control, Kinase Assay, Activity Assay, Positive Control, Negative Control