The 3a protein increases phosphorylation of IFNAR1 and its subsequent ubiquitination.

<p>(<b>A</b>) Huh7 cells were transfected with plasmid pSGI-HA or pSGI-3a-HA (2 µg each); untransfected cells were also treated with 1 µM tunicamycin for 16 hr before harvest as a positive control for ER stress. Cell lysates were prepared 48 hr post-transfection and equal amounts of proteins were subjected to immunoprecipitation (IP) using IFNAR1 antibody followed by western blotting with a phosphoserine antibody. Cell lysates were also subjected to western blotting with indicated antibodies as controls. (B) Transfections were as in (A) above. Normalized cell lysates were subjected to immunoprecipitation (IP) with an anti-IFNAR1 antibody followed by western blotting (WB) with an anti-Ubiquitin antibody. Cell lysates were also directly subjected to western blotting with indicated antibodies as controls. Arrows indicate the partially (lower band) and heavily glycosylated (higher band) forms of the IFNAR1 protein. The transfections were carried out in 60 mm dishes.