PMA-induced cytoplasmic domain phosphorylation in full-length TF. (A) IL-2RTF, TF, and cytoplasmic domain–mutated TF (Cys245Ser, TF C-S) transduced HUVECs were either incubated with 2-BP as indicated (+) immediately after transduction or were incubated without 2-BP (-). Forty-eight hours after transduction cells were either left untreated (-) or were stimulated for 1 hour with 20 ng/mL PMA (+). Cells were lysed in 2 × reducing sample buffer and Western blot analysis was performed with αP-TFCD and αTFCD antibodies. (B) TF C-S–transduced cells were preincubated with the indicated concentrations of methyl-β–cyclodextrin (cyclodextrin) or 100 nM GF109203X (GF) for 30 minutes. D indicates desensitation with 20 ng/mL PMA for 24 hours before PMA induction for the indicated times. Cells were lysed in 2 × reducing sample buffer and Western blot analysis was performed with αP-TFCD and αTF antibodies. (C) Wild-type TF and cytoplasmic domain mutants of TF (Lys255Ala, Pro259Ala, and Cys245Ser) were expressed in HUVECs. Immediately after transduction cells were incubated with 2-BP (bottom) or not treated (top) for 48 hours. After PMA induction for 1 hour, cells were lysed in 2 × reducing sample buffer and Western blot analysis was performed with αP-TFCD and αTF antibodies.