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. 2019 Apr 16;9:6169. doi: 10.1038/s41598-019-42229-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Conjugation of JOC-x with PEG-biotin or DOTA-EU. Panel (a) Shows the viral inhibition curves for JOC-x before and after conjugating to PEG2- or PEG11-biotin. Panel (b) JOC-x and JOC-Biotin conjugates were mixed with streptavidin-europium (SA-Eu) and resolved by SDS-PAGE with or without boiling or reduction by DTT. One gel was stained with Coomassie to visualize formation of multimers and one gel transferred to a PVDF membrane and imaged using a Spectramax I3x Imager to visualize Eu phosphorescence at an emission spectrum of 616 nm. Multimeric forms are indicated to the right of the gel/blot whereby SA-Eu = free streptavidin-europium, M = Monomer, T = Trimers, T-D = Trimer-Dimers, and Agg. = Aggregates/multimers. Numbering of lanes is as follows: 1 = JOC-x, 2 = JOC-PEG2-biotin, 3 = JOC-PEG11-biotin, 4 = JOC-PEG2-biotin-SA-Eu, 5 = JOC-PEG11-biotin-SA-Eu, 6 = SA-Eu alone. Panel (c) A mass spectrometry spectrum for JOC-DOTA is shown indicating the position of unconjugated JOC-x (24137 Da) and JOC-DOTA (24663 Da). Panel (d) Unconjugated JOC-x (1), JOC-DOTA (empty) (2), and JOC-DOTA (Eu) (3) were resolved by SDS-PAGE, with or without boiling or reduction by DTT. One gel was stained with Coomassie (top gel) to visualize formation of multimers and one gel transferred to a PVDF membrane and imaged using a Spectramax I3x Imager (lower blot) as described. Positions of Trimers “T” and monomers “M” are noted to the right of the gel/blot. Panel (e) Viral inhibition curves of JOC-x, JOC-DOTA (Empty), and JOC-DOTA (Eu) are shown indicating that DOTA-Eu conjugation only minimally reduces DSG2-mediated viral entry.