Thrombin-induced DAF gene transcription is independent of de novo protein synthesis.
(A) HUVECs in 75-cm2 flasks were pretreated with CHX (1 μg/mL) or plain medium alone for 30 minutes prior to the addition of thrombin (10 U/mL) and culture for up to 24 hours. Total RNA was isolated, and Northern blots were prepared. The figure shows unstimulated HUVEC (lane 1) thrombin treatment for 2 hours (lane 2), 4 hours (lane 3), 6 hours (lane 4), and 24 hours (lane 5); CHX alone for 4 hours (lane 6); and CHX and thrombin for 2 hours (lane 7) and 4 hours (lane 8). The ethidium bromide–stained gels confirmed equal loading of RNA in each lane. (B) Quantification of mRNA levels in resting and thrombin-stimulated ECs using densitometric scanning. (C) HUVECs in 75-cm2 flasks were pretreated with CHX (1 μg/mL) or plain medium alone for 30 minutes prior to the addition of VEGF (25 ng/mL) and culture for up to 24 hours. Total RNA was isolated, and Northern blots were prepared. The figure shows unstimulated HUVECs (lane 1); VEGF treatment for 3 hours (lane 2), 6 hours (lane 3), and 9 hours (lane 4); CHX alone for 6 hours (lane 5); CHX and VEGF for 6 hours (lane 6); and CHX and VEGF for 9 hours (lane 7). The ethidium bromide–stained gels confirmed equal loading of RNA in each lane. (D) Quantification of mRNA levels in resting and VEGF-stimulated ECs using densitometric scanning.
