2.3. Transient transfection and luciferase assays
Total RNA was isolated from HUVECs and cDNA was synthesized using M-MLV reverse transcriptase (Promega, Madison, WI). IL-33 cDNA (817 bp) was amplified from HUVEC cDNA and subcloned into pFLAG-CMV2. ICAM-1 (?1350 bp), VCAM-1 (?1716 bp), and 4x-κB luciferase reporter constructs were used as previously described [11]. The NF-κB p65 promoter was amplified from a BAC TAK-632 (RP11–856B14) and subcloned into pGL3-Basic (Promega). HUVECs were transfected with expression plasmid, reporter construct, and pRL-CMV for normalization using Lipofectamine as per the manufacturer’s instructions (Invitrogen). After 24 h, HUVECs were lysed with passive lysis buffer and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega).
2.4. Transfection with small interfering (si)RNA
HUVECs were transfected with control siRNA and IL-33 siRNA (Dharmacon, Lafayette, CO) using Lipofectamine for 3 h. Cells were assayed 48 h after transfection. For IL-33, siRNAs were used: 5′-GCACAUACAAUGAUCAAUC-3′. To silence ST2 expression, a gonadotropins ST2 siRNA sequence (Dharmacon; 5′-CGAAAGAGCAGGCGGCACAUU-3′) was used.
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Transient transfection and luciferase assays Total RNA was isolated
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