β-TRCP regulates VEGFR2 protein levels in HMVECs. (A) Immunoblot analysis of HMVECs stably expressing lentiviral vectors encoding shRNA specific for GFP, β-TRCP1, and β-TRCP1+2. Data shown is representative of two independent experiments. (B and C) Real-time RT-PCR analysis of β-TRCP1 (B) and β-TRCP2 (C) mRNA expression in HMVECs infected with the indicated shRNA lentiviral vectors. Error bars indicate mean ± SD from three independent experiments. ***, P < 0.001. (D) Real-time RT-PCR analysis of VEGFR2 mRNA expression in HMVECs infected with the indicated shRNA lentiviral vectors. Error bars indicate mean ± SD from three independent experiments. (E) HMVECs were infected with the indicated shRNA lentiviral vectors. After selection with 1 µg/ml puromycin for 72 h, cells were split into 60-mm dishes and, after another 20 h, treated with 20 µg/ml CHX. At the indicated time points, WCLs were prepared, and immunoblots were probed with the indicated antibodies. Data shown is representative of three independent experiments. (F) Immunoblot analysis of the HMVECs shown in A, stimulated where indicated with 100 ng/ml VEGF-A for 15 min. Data shown is representative of two independent experiments.