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Total RNA isolation and RT-PCR ------------------------------ The HUVECs seeded in 6-well plates were preincubated with or without FK506 for 4 h and then stimulated with 1 ng/mL TNF-α for 12 h. Total RNA was isolated from the HUVECs with TRIZOL (Invitrogen) according to the manufacturer's instructions. First-strand cDNA was synthesized using 2 μg of total RNA and the SuperScript™ First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer's protocol. RT-PCR was performed with the primers, sense primer, 5'-AGCCCCCAGGCTCTAACTG-3' and antisense primer, 5'-GTCGAAGGTGCTGGTGAATG-3', under the following conditions: denaturation step at 95°C for 30 seconds, annealing step at 55°C for 30 seconds, and extension step at 72°C for 30 seconds. The amplified products were separated by 1.5% agarose gel electrophoresis. 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