Comparative Functional Analysis of 12 Mammalian IFN-?4 Orthologs

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

Cells were transfected for 48 h on Nunc Lab-Tek II chamber slides (Thermo Scientific) at a concentration of 20,000 cells/ chamber in 200 mL reaction volumes. Cells were fixed for 20 min in cytofix (BD Biosciences), and then coincubated for 2 h with the MAB-IFN-l4 (2 mg/mL) in cytoperm (BD Biosciences) and rabbit a-tubulin ab (1:500 dilution, ab-15246; Abcam). Samples were then incubated for 1 h at room temperature with donkey anti-mouse Alexa Fluor 594 and donkey anti-rabbit Alexa Fluor 488 secondary antibodies (1:1000 dilutions; Life Technologies). Alternatively, Halo-tag was detected with cell-permeant Halo-tag ligand (TMR red; Promega), which was added to live cells (1:2000 for 15 min), then the cells were washed and fixed for 20 min in cytofix (BD Biosciences). Slides were covered with mounting media (Prolong Gold antifade reagent with DAPI). Immmunofluorescent images were obtained with a confocal laser scanning microscope (LSM 700; Carl Zeiss) and analyzed using Zen software (Carl Zeiss).