Figure 4
From: Cautionary notes on the use of NF-κB p65 and p50 antibodies for CNS studies
Western blots and immunohistochemistry show nonspecific protein marking by antibodies raised against phospho-ser276-p65. Antibody CS 3037 marking of proteins in gels run with extracts from hippocampal neurons in culture was exclusively nuclear, induced by 10 μM glutamate (a) or by 10 and 20 μM PMA or 20 or 30 μM forskolin (b) for 10 min. Marking of the nuclear proteins was blocked by the synthetic peptide used for immunization (2.5 μg per reaction in western blots) (c, d). However, the induced labeled proteins did not migrate at the molecular weight for p65. Immunohistochemical staining of the large molecular weight proteins (but not phospho-ser276-p65) marked by the phospho-ser276 antibody CS 3037 applied at 1:200 dilution is shown in e-g. Intrahippocampal KA injection caused a rapid (within 15 min) local neuronal response in granule cell and pyramidal neurons ipsilaterally (e, g) but not contralaterally (f, g). High-magnification views of the dentate gyrus on the left (g) and right (h) sides are taken from the boxed areas above. Preincubating the tissue with 1 μl/ml of a 1 mg/ml phosphorylated (j) or non-phosphorylated (i) p65 peptide fragment against which the antibody was generated demonstrated that only the phosphorylated peptide blocked the staining. Magnification bar in e = 200 μm for d,e,h, i. Bar in g = 20 μm for f, g.