BMPER is down-regulated in vascular inflammation. (A) Stimulation of HUVECs with indicated TNFα concentrations for 24 hours down-regulated BMPER RNA expression in a concentration dependent manner as shown by quantitative real-time PCR of at least 3 independent experiments. RNA expression was analyzed using specific primers for human BMPER and human RNA polymerase II (hRPII). (B) BMPER protein expression in HUVECs as shown by Western blotting was also decreased after incubation with TNFα (50ng/mL) for 72 hours. β-tubulin was used as loading control. Expression was quantified by densitometric analysis of 3 independent experiments (right panel: representative Western blot). (C) TNFα inhibited BMPER promoter activity in HUVECs. Cells were transfected with the respective BMPER promoter constructs and luciferase activity was quantified after 24 hours TNFα (50 ng/mL) treatment. Values represent the mean ±SD of 3 independent experiments normalized to β-galactosidase (*P < .05 versus control). (D) LPS decreased BMPER protein levels in vivo as demonstrated by Western blotting. C57BL/6 mice were injected with LPS for 3 days (2.5 μg/g/d). Lungs were minced in RIPA buffer to obtain protein. Lysates were used for Western blotting. GAPDH was used as loading control (right panel: representative Western blot).