VEGF promotes phosphorylation, ubiquitination, and degradation of IFNAR1. (A) Immunoblot analyses of VEGFR2, IFNAR1, and β-actin in HUVECs pretreated with inhibitors against PKD (CID755673, 100 μM) or PKC (Bis-I, 2 μM) for 2 hours and then treated with VEGF (100 ng/mL) for 1 hour. (B) Immunoblot analysis of phosphorylation and levels of FLAG-tagged IFNAR1 stably expressed in STAT1-deficient U3A cells that received indicated shRNA and were treated with VEGF as indicated. (C) Immunoblot analysis of ubiquitination, phosphorylation, and the levels of endogenous IFNAR1 immunopurified from HUVECs treated with VEGF as indicated. Phosphorylation and levels of PKD2 in whole cell lysates (WCL) were also analyzed. (D) Immunoblot analysis of ubiquitination, phosphorylation, and levels of Flag-IFNAR1 stably expressed in U3A cells treated with VEGF (100 ng/mL) as indicated. Phosphorylation and levels of PKD and Erk in WCL were also analyzed. (E) Cychoheximide (CHX) chase analysis of turnover of endogenous IFNAR1 in HUVECs untreated or treated with VEGF. Equal loading was verified by analysis of β-actin in these samples. The graph depicts percentage of remaining IFNAR1 at the indicated time points. (F) Degradation of Flag-IFNAR1 in U3A cells treated with VEGF (100 ng/mL) and CHX (20 μg/mL) as indicated.