FIG. 3.

The IFNaR2 ECD is shed from live cells. (A) Schematic of GFP-IFNaR2 construct. The ECD, TMD, ICD, and HA epitope tag are indicated. (B) Immunoprecipitation of the shed ECD from the culture medium. HEK293T cells were transfected with plasmids encoding GFP or GFP plus the construct shown in (A). Lane 1 is a control immunoprecipitation of lysate from GFP-transfected cells. In lanes 2 and 3, the cell culture medium was immunoprecipitated with the indicated antibodies and immunoblotted with anti-GFP. The GFP signal in lane 2 represents spill-over from lane 1. (C) The recovered IFNaR2 ECD is N-glycosylated. Immunoprecipitates prepared as in (B) were incubated with 1 U PNGase F at 37°C for 1 h and then immunoblotted with anti-GFP (upper portion of filter) or anti-rabbit IgG (lower portion of filter). (D) Full-length IFNaR2 is similarly N-glycosylated. Lysates from HEK293T cells transfected with the construct shown in Fig. 1A were incubated with PNGase F for 0, 1, or 4 h and immunoblotted with anti-HA. These immunoblots are representative of 2 replicates. PNGase F, protein N-glycosidase F; GFP; IP; HA.