Figure 2

IκBα deletion results in increased NFκB activity in astrocytes. (A and B) Representative images of wild-type (WT) and IκBα knockout (KO) primary neurons, either under basal condition (A) or treated with 50 ng/ml TNFα for 30 min (B), followed by staining against neuronal marker Neuronal nuclei (NeuN) and NFκB subunit p65, and displayed as individual or merged images. Note the uniform p65 staining in NeuN-positive neurons under both conditions and the cells that undergo p65 nuclear translocation in response to TNFα (B) are NeuN-negative. (C and D) Representative images of primary astrocytes, either under basal condition (C) or treated with TNFα (D), followed by staining against astroglia marker GFAP and p65, and displayed as individual or merged images. Note clear p65 nuclear translocation in GFAP-positive astrocytes. (E) Representative p65 immunostaining of primary WT and KO astroglia cultures. (F) Quantification of relative p65 nuclear to cytoplasmic intensity in WT and IκBα KO neurons with or without TNFα. (G) Quantification of relative p65 nuclear versus cytoplasmic fluorescence intensity in WT and IκBα KO astrocytes in the presence or absence of TNFα stimulation, documenting increased basal p65 in IκBα KO sample and greatly enhanced nuclear p65 upon TNFα treatment in both WT and KO cultures. N = 7-10/genotype. (H) p65 ELISA quantification of nuclear preparations from adult (2–3 month) Ctrl and IκBα astroglia-specific knockout (GcKO) hippocampal samples. N = 3/genotype. ns: non-significant; *p < 0.05. Scale bar: 25 μm.