Comparative Functional Analysis of 12 Mammalian IFN-?4 Orthologs

Paquin, Ashley; Onabajo, Olusegun O.; Tang, Wei; Prokunina-Olsson, Ludmila

Next, we tested the possibility of detection of IFN-l4 orthologs with two custom monoclonal antibodies generated against nonoverlapping peptides within the N and Cterminal parts of the human IFN-l4 protein. In Western blot analysis (Fig. 3) , flow cytometry ( Fig. 4 and Supplementary Figs S1 and S3), and confocal imaging (Fig. 5) , the mouse monoclonal antibody (MAB-IFN-l4) recognized IFN-l4 from the human, chimpanzee, orangutan, rhesus, marmoset, cynomolgus (all the primates in the panel), and elephant. The results were similar for orthologs that were detectable both by Halo-tag and MAB-IFN-l4. The rabbit monoclonal antibody (RAB-IFN-l4) recognized only the human IFN-l4. This is likely because the area that can be recognized by MAB-IFN-l4 is more conserved between species compared with the area that can be recognized by the RAB-IFN-l4 (Fig. 1) . Further genomic studies may help to identify IFN-l4 orthologs in other relevant species. The success and accuracy of these predictions rely on the quality and completion of available genomic sequencing and additional targeted DNA sequencing is often needed to validate the existing sequences and close the gaps. Detailed analysis of functional properties of IFN-l4 orthologs in relation to differences in protein sequences could provide additional information on its biology.