4.12. NF-κB Activity Assay

The NF-κB activities in HUVECs were measured using luciferase activity protocols [42]. In brief, HUVECs incubated in a six-well plate to reach 80% confluency were co-transfected with Renilla reniformis luciferase reporter vector (Promega, Madison, WI, USA) and NF-κB-driven luciferase vector (Stratagene, La Jolla, CA, USA) at a ratio of 2:5 using Lipofectamine (Invitrogen, Carlsbad, CA, USA). After treatment with 10 nM α-MSH either/or combination with 10 ng/mL LPS, 10 μg/mL MC1-R antibody, 10 μg/mL MC2-R antibody, MC1-R and MC2-R siRNA for 24 h, the luciferase activities were detected with Dual-Light kits (Promega, Madison, WI, USA) and the luminometer (Microlumat Plus LB96V; Berthold Technologies, Bad Wildbad, Germany) and normalized with that of R. reniformis luciferase, according to the manufacturer’s instructions.