Mammalian expression system

Three to five clones of each IFN subgroup were selected by PCR and sequencing confirmation. Two or three clones with the highest sequence identity (98–100%) to the template sequence extracted from Xenbase genomic sequences were used for functional studies. Endotoxin-free plasmids (equal to 0.25 μg/well in 96-well cell culture plates) from confirmed clones were purified from individual bacteria cultures and used for cell transfections in A6 frog cells for transient expression and antimicrobial assays10 (link)48 (link)51 (link)53 (link). For peptide production, IFN-expression plasmids were transfected in HEK293F cells of a mammalian-expression system (Invitrogen) to produce IFN peptides as previously described10 (link). Antimicrobial assays were conducted as described10 (link)48 (link)51 (link)53 (link). Induction of IFN-stimulating genes (ISGs), exemplified for both robust and turntable ISGs, was determined in cells treated with indicated amphibian IFN peptides (20 ng/ml) in 24-well cell culture plates10 (link)48 (link)51 (link)53 (link).

ADGRL4/ELTD1 overexpression was performed by transducing low passage HUVECs with an ADGRL4/ELTD1 containing lentivirus using the Mammalian Expression System with Gateway Technology (Invitrogen) as per the manufacturer’s protocol. An empty lentivirus was used as the control. Cells were assayed for overexpression using qPCR.