Phosphorylation of the TF cytoplasmic domain in the absence of the autologous extracellular domain requires Golgi sorting. (A) Endothelial cells were untransduced (ctrl) or transduced 48 hours earlier with IL-2R, IL-2RTF, or IL-2RTFAA. Cells were stimulated with 20 ng/mL PMA for 1 hour and lysed with sample buffer for Western blotting with 1 μg/mL phosphorylation-specific anti-TF cytoplasmic domain antibody (αP-TFCD) and with αIL-2R antibody as loading control. (B) Time course experiment of PMA-stimulated endothelial cells expressing IL-2RTF. Loading was controlled by Western blotting with αTFCD antibody to the unphosphorylated TF cytoplasmic domain. (C) Disruption of Golgi transport by BFA prevents TF cytoplasmic domain phosphorylation. Note the difference in glycosylation of IL-2RTF in BFA-treated cells. (D) Phosphorylated TF cytoplasmic domain is expressed on the apical surface of endothelial cells. IL-2RTF–transduced HUVECs were stimulated with PMA for the indicated times. After surface biotinylation at 4°C, IL-2RTF was sequentially precipitated with αIL-2R antibody and streptavidin beads followed by Western blot analysis with αIL-2R or αP-TFCD antibodies. (E) PMA-induced phosphorylation of the TF cytoplasmic domain is dependent on PKCα and membrane composition. Cells were pretreated with the indicated concentrations of the cholesterol-depleting reagent methyl-β–cyclodextrin (cyclodextrin) or with the indicated kinase inhibitors for 30 minutes. PKC inhibitors were used at the following concentrations: 100 nM GF109203X (Gf), 1 μMGö6976 (G1), 6 μM Rottlerin (Ro), 2 μM Hispidin (H), 1 μMGö6983 (G2), and different concentrations of Safingol at 50 μM (Sl) or 200 μM (Sh). After PMA induction for 1 hour, cells were lysed in 2 × reducing sample buffer and Western blot analysis was performed with αP-TFCD and αIL-2R antibodies. In the middle panel, αTFCD antibody was used as loading control instead of αIL-2R.