Summary of anti-EBNA1 murine mAbs.

<p>WB: Western blot, WB': Western blot signal with endogenous EBNA1, ELISA: the average ELISA signal when adding equal concentrations of purified mAb to an EBNA1-coated well, PR-mAb: polyol-responsive mAb, IP: immunoprecipitation, Supershift: ability to bind to EBNA1/DNA complex and change its mobility during non-denaturing gel electrophoresis, NYT: not yet tested. Epitope was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004614#pone-0004614-g001" target="_blank">Fig. 1</a>.</p>*<p>particularly high affinity for endogenous EBNA1.</p>a<p>responsive to salt alone.</p

Immunoprecipitation of EBNA1.

<p><i>E. coli</i>-expressed EBNA1 (∼50 kDa) was isolated from whole cell extract by mixing with equal quantities of mAb-rProteinA-agarose resin. Reactions were allowed to incubate for 1 h, washed to remove nonspecifically bound proteins, and eluted with SDS sample buffer. MW is molecular weight. The Western blot was probed with rat mAb 2B4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004614#pone.0004614-Grasser1" target="_blank">[23]</a>.</p

Polyol-responsiveness of anti-EBNA1 mAbs.

<p>A) ELISA-elution assay results to determine polyol-responsiveness. A standard ELISA protocol was performed with the addition of an intermediate step. After the anti-EBNA1 mAbs are bound to the EBNA1-coated well, either a control buffer (TE: 50 mM Tris-HCl, pH 7.9+0.1 mM EDTA) or a salt-polyol buffer (AS/PG: TE buffer+1 M ammonium sulfate+40% propylene glycol or NaCl/PG: TE buffer+1 M NaCl+40% propylene glycol) was added to the wells. MAbs that are polyol-responsive will have lower affinity for their antigen in the salt-polyol buffer which will result in a decrease in signal after the addition of a secondary antibody and the ELISA substrate. The experiment was done in triplicate. B) Detailed analysis of mAb 3EB7 response to various combinations of ammonium sulfate (AS) and propylene glycol.</p

Immunoaffinity chromatography using PR-mAbs.

<p>The soluble fraction of whole cell extract from <i>E. coli</i> overexpressing EBNA1 was added to A) mAb 1EB2-Sepharose column and B) mAb 3EB7-Sepharose column, washed, and eluted with TE+0.75 M AS+40% PG. Coomassie blue stained gels are shown.</p

Ability of mAbs to bind EBNA1/DNA complex.

<p>MAbs were tested by EMSA to determine binding efficiency to a pre-formed EBNA1/DNA complex. Purified EBNA1 protein was bound to a ³²P-labelled oligonucleotide encoding one palindromic EBNA1 binding site. Equal concentrations of purified mAb were added to each reaction and allowed to incubate. The ability of the mAbs to shift the EBNA1/DNA complex was analyzed by gel electrophoresis. The first lane does not include any protein. The second lane contains only DNA and EBNA1. MAbs are listed by the last number in their name.</p

Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors

Novel engineered NanoLuc (Nluc) luciferase being smaller, brighter, and superior to traditional firefly (Fluc) or <i>Renilla</i> (Rluc) provides a great opportunity for the development of numerous biological, biomedical, clinical, and food and environmental safety applications. This new platform created an urgent need for Nluc inhibitors that could allow selective bioluminescent suppression and multiplexing compatibility with existing luminescence or fluorescence assays. Starting from thienopyrrole carboxylate <b>1</b>, a hit from a 42 000 PubChem compound library with a low micromolar IC₅₀ against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nanomolar, cell permeable inhibitors. Further elaboration revealed a channel that allowed access to the external Nluc surface, resulting in a series of highly potent cell impermeable Nluc inhibitors with negatively charged groups likely extending to the protein surface. The permeability was evaluated by comparing EC₅₀ shifts calculated from both live and lysed cells expressing Nluc cytosolically. Luminescence imaging further confirmed that cell permeable compounds inhibit both intracellular and extracellular Nluc, whereas less permeable compounds differentially inhibit extracellular Nluc and Nluc on the cell surface. The compounds displayed little to no toxicity to cells and high luciferase specificity, showing no activity against firefly luciferase or even the closely related NanoBit system. Looking forward, the structural motifs used to gain access to the Nluc surface can also be appended with other functional groups, and therefore interesting opportunities for developing assays based on relief-of-inhibition can be envisioned