NF-κB Protein Analysis by Western Blot
Variable analysis
- NF-κB Western blotting was carried out as reported previously (18 (link))
- Levels of NF-κB, phospho-NF-κB, IκB, and phospho-IKB kinase proteins
- Whole cell lysates were isolated with Halt™ Protease and Phosphatase inhibitors (Thermo Fisher) included in RIPA buffer (Thermo Fisher) during lysis
- Nuclear and cytoplasmic extracts were prepared using a Nuclear Extract kit (Active Motif)
- Protein was quantified using a Pierce™ BCA protein assay (Thermo Fisher), after which all samples were adjusted to the same concentration
- Samples were loaded in lanes of pre-cast 4-20% Mini-Protean TGX Protein Gels (Bio-Rad) and electrophoresis performed at 100V for 90 min in a BioRad mini-PROTEAN tetra vertical electrophoresis chamber
- Proteins were transferred to a low fluorescence nitrocellulose membrane (Bio-Rad) using the Bio-Rad TransBlot Turbo System, per the manufacturer's instructions
- Antibody binding was performed using an iBind Flex apparatus per the manufacturer's instructions
- Rabbit anti-Actin (Cell Signaling Technologies, 1:4000)
- Not explicitly mentioned
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