Pcmv gfp

The endothelial cells HUVEC and BAEC were grown at 70–80% confluent and used between passages 3 and 8 as previously reported [20] (link), [52] (link), [53] (link). Infection with adenovirus encoding OGT, OGA, or GFP (as adenoviral infection control), as described previously [45] (link), [46] (link). Confluent endothelial cells were infected with transfection-ready plasmid encoding Ub^G76V-GFP plasmid as previous reported [48] (link). It is used as well as their control DNA plasmids (pCMV-GFP) according to instructions provided by OriGene (Rockville, MD). Transfection of control or target (OGT) siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA) as described previously [20] (link). All cells were incubated in a humidified atmosphere of 5% CO₂ at 37°C.

The plasmids pCMV-CYP2C8 and pCMV-GFP were obtained from OriGene Technologies Inc. (Rockville). As the density of the cultured cells reached ~50–70%, each group was pretreated with Lipofectamine 2000 and then interfered by adding the pCMV-CYP2C8 and pCMV-GFP plasmids, respectively. After 6 h of transfection, the experimental medium was added to the cells followed by exposure to TNF-α (10 ng/ml) in the absence or presence of GW9662 (1 μM). The efficacy of transfection was obtained by examining the green fluorescence by microscopy (Nikon, Tokyo, Japan).