Expression of KLF2 or KLF4 inhibits NF-κB transcriptional activity in APLA/anti-β2GPI–treated endothelial cells. (A) Endothelial cells were transfected with expression vectors containing scrambled DNA (Scr DNA, control), or KLF2 or KLF4 cDNA along with an NF-κB–luciferase reporter and Renilla luciferase (as a transfection efficiency control). Transfected cells were subsequently incubated with medium alone, β2GPI (100nM) and anti-β2GPI antibodies (600nM), or TNF-α (10 ng/mL) for 5 hours before measurement of luciferase activity. NF-κB transcriptional activity was determined from measured NF-κB luciferase activity normalized to Renilla. Expression of KLF2 and KLF4 inhibited NF-κB transcriptional activity in the presence of β2GPI/anti-β2GPI antibodies compared with control cells (**P < .004 and *P < 0.04, respectively). Error bars represent the mean ± SEM of triplicate points, and data are representative of 4 experiments. (B) KLF2 expression does not inhibit phosphorylation of p65 serine 536. Endothelial cells were transfected with expression vectors containing scrambled DNA (Scr DNA, control), or KLF2 cDNA and subsequently treated as in panel A. Cell extracts were prepared, and 80 μg of total protein was separated using 7.5% SDS-PAGE, transferred to PVDF, and blotted with rabbit anti–human antibodies to phospho-p65 (serine 536) and total p65. (C) KLF2 expression does not block nuclear translocation of NF-κB p65. Endothelial cells were transfected and treated as in panel B. Nuclear (Nuc) and cytoplasmic (Cyt) extracts were prepared, and 40 μg of protein from each was separated by 7.5% SDS-PAGE, transferred to PVDF, and blotted with rabbit anti–human p65. Transfection of endothelial cells with a KLF4 expression vector yielded identical results as seen with KLF2 in panels B and C.
