MUC5AC drives COPD exacerbation severity through amplification of virus-induced airway inflammation

Aran Singanayagam; Joseph Footitt; Benjamin T Kasdorf; Matthias Marczynski; Michael T Cross; Lydia J Finney; Maria-Belen Trujillo Torralbo; Maria Calderazzo; Jie Zhu; Julia Aniscenko; Thomas B Clarke; Philip L Molyneaux; Nathan W Bartlett; Miriam F Moffatt; William O Cookson; Jadwiga Wedzicha; Christopher M Evans; Oliver Lieleg; Patrick Mallia; Sebastian L Johnston

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/706804 doi: bioRxiv preprint Visualization of mucin-associated DNA Successful removal of DNA from MUC5AC was verified by electrophoretic separation of the mucin samples and subsequent visualization of associated DNA molecules via the DNAbinding dye SYBR Green I (S9430, Sigma Aldrich, St. Louis, MO, USA). Therefore, 40 µg of the mucin samples were mixed with Laemmli buffer, loaded on a polyacrylamide gel (Mini-Protean TGX Gels 4-20%, #45-1093, BioRad, Hercules, CA, USA) and separated (60 V for 10 min and 120 V until the dye front reached the end of the gel). The gel was stained using SYBR Green I afterwards (diluted 1:10.000 in TBE buffer) at room temperature under gentle shaking for 30 min. The DNA stain was visualized with a Gel Doc EZ Gel Documentation System (Biorad) with epi-illumination at a wavelength of 254 nm (Fig. 1) . We additionally confirmed that DNA was undetectable in samples of MUC5AC protein using Quant-iT PicoGreen dsDNA reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol, as previously described 8 .