FIGURE 4.

PSD-95 protein and mRNA levels are elevated in Egr-1 KO mouse hippocampi. Hippocampi of WT and KO mice (each 6 months old) were analyzed by Western blotting, qPCR, and immunohistochemistry. A, levels of synaptophysin and N-cadherin are not altered in Egr-1 KO mouse hippocampus. Hippocampal extracts of adult WT and Egr-1 KO mice (n = 3 in each group) were analyzed by quantitative Western blot analysis. Based on band intensities, relative amount of each protein was determined and compared. Upper panel is a representative Western blot. Lower panel indicates quantification data from three animals in each group. B, quantitative Western blot and qPCR analyses for PSD-95. Upper panel shows a representative Western blot. Middle panel is the result of Western blot quantification. Data presented are from three animals from each group. *, p < 0.05 (n = 3) with respect to WT. Lower panel shows quantitative RT-PCR analysis of PSD-95 mRNA (normalized to β-actin) in hippocampus of KO and WT genotypes (see Table 1 for primers used). The data are from three mice in each group. *, p < 0.04 with respect to WT. C, immunohistochemistry. Upper panel is a representative micrograph. Middle panels are corresponding insets at higher magnification. Immunohistochemical staining revealed widespread expression of PSD-95 in the hippocampal formation in both genotypes. Compared with WT, KO mice show higher PSD-95 immunostaining in CA1, CA2, and DG subfields. Scale bars, 100 μm in upper panel and 20 μm in middle panels. Lower panel shows analysis of PSD-95 immnoreactivity (optical density) in the CA1 and CA2 of hippocampus. Quantification of immunohistochemical staining was performed using the Spectrum Analysis algorithm package and ImageScope analysis software (version 11.2, Aperio Technologies). A rectangular region of interest, 25 μm² in size, was defined. Immunoreactivity value for each region of interest was obtained. The immunoreactivity within each regions of interest in each section was averaged to generate a mean immunoreactive value for each animal. Data were averaged from three mice each with four regions (n = 12) for each genotype and are expressed as the fold of WT. ***, p < 0.001 (n = 3) with respect to WT.