TF cytoplasmic domain–mediated apical sorting in endothelial cells is independent of phosphorylation. (A) IL-2R, IL-2R fused with wild-type TF cytoplasmic domain (IL2RTF), IL-2R chimeras with Ser253/258 mutation to Ala in the TF cytoplasmic domain (IL2RTFAA), and IL-2R chimeras with Ser253/258 mutation to Asp in the TF cytoplasmic domain (IL2RTFDD) were transduced by adenovirus into HUVECs and protein expression after 48 hours was detected by Western blotting with αIL-2R antibody. Untransduced cells (ctrl) do not express the murine IL-2R. (B) High–molecular weight (Golgi matured) versus low–molecular weight (unmodified) protein was quantified by densitometry and blotted as percentage of Golgi-matured protein. Mean and standard deviation from 3 independent experiments is shown. (C) Mobility differences of IL-2R and IL-2RTF are due to N-linked glycosylation. Glycosylation was blocked by incubation with 10 μg/mL tunicamycin (TUN) or 10 μg/mL brefeldin A (BFA) for 48 hours. IL-2R in untreated (ctrl) and treated cells was detected with αIL-2R antibody. (D) Deglycosylation of immunoprecipitated protein with Endo H or N-glycosidase F (PNG F) was followed by Western blotting with αIL-2R antibody. (E) IL-2R–and IL-2RTF–transduced HUVECs were apically biotinylated and immunoprecipitated with αIL-2R antibody (7G7B6). Efficiency of precipitation was determined by Western blotting with αIL-2R (left) and biotinylation was detected by streptavidin-HRP (SA-HRP, right). (F) IL-2R–transduced cells were biotinylated as monolayer (ADH) or in solution (SOL), immunoprecipitated and resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) for subsequent Western blot analysis with αIL-2R (left) and SA-HRP (right).