Bats Possess Unique Variants of the Antiviral Restriction Factor Tetherin

Joshua A. Hayward; Mary Tachedjian; Adam Johnson; Tamsin B. Gordon; Jie Cui; Glenn A. Marsh; Michelle L. Baker; Lin-Fa Wang; Gilda Tachedjian

To visualise tetherin localization within cells, 500 ng of plasmids encoding HA-tagged human l-tetherin and P. alecto tetherin isoforms A, B, and C were transfected into HEK293T cells seeded on glass coverslips as described above. At 48 h post transfection, cells were fixed with . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.08.031203 doi: bioRxiv preprint 4% paraformaldehyde (Sigma) in PBS for 10 min at room temperature, and then permeablised in 0.2% Triton X-100 in PBS for 5 min at room temperature. Tetherin localisation in cells was detected by staining cells with anti-HA-tag rabbit monoclonal IgG (Thermo) diluted in 0.2% Triton X-100, 3% BSA in PBS for 1 h at room temperature. Cells were subsequently stained with anti-rabbit AlexaFlour 488 secondary IgG (Thermo) diluted in 0.2% Triton X-100, 3% BSA in PBS for 20 min at room temperature. Nuclei are blue stained with Hoechst 33342 (Thermo) according to the manufacturer's instructions. Coverslips were mounted onto glass slides with ProLong Gold Antifade Mountant (Thermo), then imaged on a Nikon AR1 confocal microscope and analysed with ImageJ (NIH) software. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.08.031203 doi: bioRxiv preprint