Nuclear factor kappa B (NF-κB) activation assay

TB Tetyana P. Buzhdygan
CR Cassidy R. Rodrigues
HM Hannah M. McGary
JK Jana A. Khan
AA Allison M. Andrews
SR Scott M. Rawls
SR Servio H. Ramirez
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To examine the activation of NF-κB subunits, hBMVEC were grown to confluency, medium was changed to growth factor-free/serum-free medium, and mephedrone was added at 10 μM final concentration for 24 h. For TLR4 inhibition experiment, cells were pre-treated with 100 nM TAK242 in growth factor-free/serum-free medium for 3 h, followed by mephedrone treatment. Confluent cell monolayers were briefly rinsed with PBS and processed for cell fractionation using ProteoExtract Subcellular Proteome Extraction kit (EMD Millipore) as per the manufacturer’s protocol. Nuclear extracts were analyzed using TransAM NF-κB Activation Assays (Active Motif, Cat No 43296) as per the manufacturer’s protocol. For analysis, 10 ug of nuclear extract was loaded per each well.

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