vGPCR and vFLIP proteins regulate Notch pathway through ERK and NF-κB, respectively. (A) HUVECs stably transfected with vFLIP-ER^TAM plasmid or control vector²³  were grown on 6-well plates and induced by 4-OHT (100nM) for 48 hours. The cells were treated with DMSO or Bay (10μM) for 2 hours. Cells without 4-OHT induction were also treated with Bay (10μM) for 2 hours before harvest. RNA was isolated and subjected to quantitative RT-PCR. Expression of Dll4, Jagged1, and Hey1 was analyzed. (B) Cells were treated in the same way as in panel A and subjected to Western blot for Dll4 and Jagged1. (C) 293T cells were transfected with vGPCR plasmid for 72 hours. Before harvest, the vGPCR-transfected cells were treated by PI3K inhibitor LY (LY294002, 15μM), ERK inhibitor U0126 (15μM), and NF-κB inhibitor Bay (Bay 11-7085, 10μM), or a combination of the 3. Cells were treated by LY and U0126 for 5 hours and Bay for 2 hours. RNA was isolated and subjected to quantitative RT-PCR. Expression of Dll4, Jagged1, and Hey1 was analyzed.