Endothelial cell activation induced by APLA/anti-β₂GPI antibodies decreases expression of KLF2 and KLF4. (A) Cells were incubated with medium alone (control), β₂GPI (100nM)/anti-β₂GPI antibodies (600nM), or TNF-α (10 ng/mL) for 5 hours. Total RNA was then isolated and KLF2 and KLF4 expression analyzed by quantitative real-time PCR. β₂GPI/anti-β₂GPI antibodies and TNF-α reduced KLF2 mRNA 5-fold compared with control (**P < .001 for both), whereas KLF4 mRNA levels were reduced 3.8-fold in the presence of β₂GPI/anti-β₂GPI antibodies (***P < .0001) but increased 2.6-fold by TNF-α (*P = .0012). Error bars represent the mean ± SEM of triplicate points. (B) Immunoblotting for KLF4 protein. Cells were treated as in panel A. Extracts were prepared and total protein (80 μg) separated using 7.5% SDS-PAGE, transferred to PVDF, and probed with goat anti–human KLF4 antibodies.