de Wolf, C.J., Cupers, R.M., Bertina, R.M. and Vos, H.L.
Notes: The Protein S promoter (PROS1) fragment –5948/–1 was cloned directly 5’ to the firefly luciferase reporter gene in the pGL3-Basic Vector using the KpnI and XhoI enzyme sites. This construct, PS5948-luc, was linearized with KpnI and NdeI and subsequently subjected to progressive deletion using the Erase-a-Base® System. The size of the resulting 5’-deletion was determined by sequence analysis, and the deletion constructs were used for transient transfection assays. HepG2, HuH7, HeLa and HUVEC cells were transfected at 60–80% confluency in 12-well plates using 3µl of Tfx™-20 per microgram DNA. In each transfection, an equimolar concentration of construct was used and supplemented with an additional plasmid to keep the amount of transfected DNA constant. pRL-SV40 Vector was co-transfected as a transfection control using a 1:500 ratio to the total transfected amount of DNA in HepG2, HuH7 and HeLa cell lines, and a 1:100 ratio in transfections with HUVEC and 1 × 106 Meg01 suspension cells. Transcription factor expression vector (250ng) was co-transfected, and expression vector without the transcription factor cDNA was used as a negative control. Cell extracts were harvested at either 24 (HepG2 and HuH7) or 48 hours (Meg01, HUVEC, and HeLa) post-transfection using 250µl of Passive Lysis Buffer per well. Luciferase activity was determined using 20–100µl of lysate with the Dual-Luciferase® Reporter Assay System. (3510)