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. 2012 Aug 30;40(20):10287–10301. doi: 10.1093/nar/gks798

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© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — NONO binds PAR in vitro. (A) SDS-PAGE of 100 ng purified His-NONO protein stained with Coomassie blue (lane 2). (B) In vitro PAR-binding assay. 1 µg of purified His-NONO was loaded on an SDS-PAGE, blotted onto a nitrocellulose membrane and incubated in 250 nM ³²P-labeled PAR in TBS-T without (lane 1), with 1-fold (lane 2), 10-fold (lane 3) or 100-fold unlabeled competitor PAR (lane 4). (C) A PAR-binding assay was conducted as in (B) without (lane 1), with 1-fold (lane 2), 10-fold (lane 3) or 100-fold unlabeled competitor RNA (lane 4). (D) Kinetics of PAR binding to purified His-tagged NONO conducted by SPR spectroscopy. To analyse binding kinetics, PAR was injected at three different concentrations (125, 250 and 500 nM). PAR injection was done for 300 s and dissociation data were collected for 600 s. Data were fitted with Langmuir 1:1 interaction plot to calculate rate constants. The sensorgram is representative of three independent experiments.