Figure 2

Western blots show p65 antibodies that failed the test of specificity in p65 KO tissues. a. The sc-8008 antibody showed staining at approximately the correct molecular weight, but it was present also in p65 KO tissues. Curiously, in protein from cultured neurons, the protein from KO cells was lacking. Protein load was 40 μg for brain, spleen and liver and 10 μg for neurons for both a and b. b. The MAB3026 monoclonal antibody against the p65 NLS showed a single band that was present in both WT and KO tissue. c. The CS 3037 antibody is raised against the phosphorylated (at Ser276) form of p65. The staining was almost entirely in the nuclear (N) fraction, absent in the cytosolic (C) fraction, and was induced in the high molecular weight range by glutamate (Glut) treatment (100 μM, 10 min) relative to unstimulated control (Cont). However, the bands were not present at the expected molecular weight for phospho-p65. Protein (5 μg per lane) is from cortical neurons. In d-g, proteins from cultured neurons were run to compare sc-372 and MAB3026. Compared with the band stained with sc-372 (d), the band stained with MAB3026 runs in the gel at a slightly higher molecular weight (e). The same gel was probed for MAB3026 (e), then stripped and re-probed for both antibodies simultaneously (f), or probed initially with both antibodies (g) to show that the single band marked by the MAB3026 antibody is not the p65 epitope.