NF-κB Western blotting was carried out as reported previously (18 (link)). Briefly, 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. The following primary antibodies used at the indicated dilution: Rabbit anti-Actin (Cell Signaling Technologies, 1:4000), NF-κB (CST, 1:1000), phospho-NF-κB (CST, 1:1000), IκB (CST, 1:1000), phospho-IKB kinase (CST, 1:1000). The following Licor (Lincoln, Nebraska) near infrared fluorescent secondary antibodies were used: IRDye® 680LT (1:4000) and IRDye® 800CW (1:3000). Blots were read using a Licor Odyssey Imaging System.
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