NEDD4 ubiquitinates IFITM3 in vitro.

<p>HA-hIFITM3 was added to reactions containing NEDD4-compatible E1 and E2 ubiquitin ligases, ubiquitin (WT or mutants in which only K48 or K63 were not mutated), and reaction buffer containing ATP in the presence or absence of NEDD4. The reaction was allowed to proceed for 1 h at 37°C, and IFITM3 was re-immunoprecipitated and subjected to Western blotting with anti-ubiquitin (Ub) and anti-HA antibodies. IgG H.C. indicates detection of the heavy chain of the immunoglobulin used for immunoprecipitation.