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.
Copyright and License information: The Author(s) ©2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this
article to respond.