Interferon-induced transmembrane protein 3 blocks fusion of sensitive but not resistant viruses by partitioning into virus-carrying endosomes

Suddala, Krishna C.; Lee, Christine C.; Meraner, Paul; Marin, Mariana; Markosyan, Ruben M.; Desai, Tanay M.; Cohen, Fredric S.; Brass, Abraham L.; Melikyan, Gregory B.

The p24 content of viral stocks was determined by ELISA, as described previously [78] . Whole cell lysates were harvested in RIPA Buffer (Sigma) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche), incubated on ice for 10 min, and cleared by centrifugation at 16,000xg for 5 min. Total protein was measured using a bicinchoninic acid assay (BCA, Pierce) and normalized protein was loaded onto 4-15% polyacrylamide gels (Bio-Rad, Hercules, CA). Precision Plus Protein Standards (Kaleidoscope Bio-Rad) were used as molecular weight markers. Proteins were transferred onto a nitrocellulose membrane, blocked in 10% Blotting-grade Blocker (Bio-Rad) in PBS-T (phosphate buffered saline with 0.1% Tween-20) for 30 min at room temperature. Membranes were incubated in primary antibodies overnight at 4ËšC in 5% Blotting-grade Blocker with gentle shaking: rabbit anti-IFITM3 (1:500), mouse anti-tubulin (1:3000), human HIV-IG) (1:2000) , and rabbit anti-WSN Influenza R2376 (1:100). After washing membranes with PBS-T at room temperature, Horseradish peroxidase-conjugated (HRP) goat anti-rabbit, rabbit anti-mouse, and goat anti-human secondary antibodies were added in 5% Blotting-grade Buffer for 1 h at room temperature with gentle shaking. Following PBS-T washing of membranes, ECL Prime chemiluminescence reagent (GE Healthcare) was used for protein detection.