Heme and LPS induce EC NF-κB activation via TLR4 signaling. (A) HUVECs were incubated with LPS (10 ng/ml), TNF-α (10 ng/ml), heme (10 µM), or TAK-242 (400 nM) + heme for the indicated times (1-8 hours). At the end of the incubation period, nuclear extracts were prepared, run on a western blot, and stained for NF-κB phospho-p65. (B) mPVECs from TLR4^+/+ or TLR4^−/− mice were incubated with heme (10 µM), LPS (10 ng/ml), TNF-α (10 ng/ml), or TAK-242 (400 nM) + heme (10 µM) for 1 hour. At the end of the incubation period, nuclear extracts were prepared, run on western blot, and stained for NF-κB phospho-p65.