E3 Ubiquitin Ligase NEDD4 Promotes Influenza Virus Infection by Decreasing Levels of the Antiviral Protein IFITM3
Fig 1
IFITM3 is ubiquitinated by NEDD4.
A) Alignment of IFITM3 N-terminal amino acids from various species. Bold and underlined text highlights the conserved PPxY motif. B) Mouse embryonic fibroblasts (MEFs) were stimulated overnight with IFN-α (160 units/mL) to ensure production of IFITM3, and imaged by fluorescent confocal microscopy with staining for endogenous IFITM3, NEDD4, LAMP1, and nuclei (DAPI). Images were taken with a 60x objective and 2.5x zoom. Pseudocolored merged images in different staining combinations are shown. C-E), HEK293T cells were co-transfected with plasmids expressing IFITM3 and epitope tagged ubiquitin ligases, NEDD4, CLB-B, SMURF1 and SMURF2, as indicated. Cell lysates were immunoprecipited with anti-myc resin, and examined by Western blotting with anti-myc and anti-ubiquitin (Ub) antibodies. Western blots of cell lysates with anti-HA (C) or anti-FLAG (D,E) antibodies were performed to confirm expression of the ubiquitin ligases. Anti-GAPDH Western blotting was performed to confirm comparable protein loading.