A number of rather moderately labeled neurons were also encountered in the bed nucleus of the stria terminalis, nucleus basalis of Meynert, globus pallidus, entopeduncular nucleus and ventral pallidum (Fig. and Barlowe, 2001; Springer et al., 2000). In the nematode reducing the activity of the p24 family member SEL-9 increased the cell surface accumulation of a transport-defective mutants of the Notch homologues, GLP-1 and LIN-12 (Wen and Greenwald, 1999). In the fly raised against stanniocalcin 2 (Ito et al., 2004) was used as negative control to establish specificity for the co-immunoprecipitation. For quantification, the immunoblots were developed by chemiluminescence method and exposed to X-ray films for various lengths of time Eptapirone to ensure that the signals are not saturated. Optimal exposures were quantified using standard densitometry and a calibration step tablet was used to convert raw optical densities to relative fold difference in signal intensity essentially as described (http://rsb.info.nih.gov/ij/docs/examples/calibration/) using Metamorph software (Molecular Devices Corporation, Downingtown, PA). Normalized signal intensities were compared between sporadic AD (mean age 64.13 3.2) and FAD cases (mean age 38.5 1) and their respective age-matched controls (older Eptapirone controls mean age 69.2 5.1 and young controls mean age 35 5, respectively). The data are presented as mean S.E.M, and statistical significance was analyzed by indicates p23 co-immunoprecipitated by PS1 antibody. (C) Immunoblot analysis of p23 in different regions of the adult rat brain. The blot was reprobed with an antibody against -actin as the loading control. Subcellular localization of p23 In cultured non-neuronal cells p23 predominantly resides in Cis-Golgi cisternae and adjacent tubulovesicular membranes (Blum et al., 1999; Rojo et al., 1997). In agreement with these previous findings, the p23 antibody employed in our investigation stained the Golgi apparatus in HeLa cells, where it colocalizes with the Golgi-resident enzyme N-acetylgalactosaminyltransferase-2 (Fig. 2A). In addition to the predominant Golgi localization, p23 staining of small vesicles is also observed. Next, we examined the localization of p23 in primary mouse cortical neuronal cultures. Consistent with p23 localization in non-neuronal cells, we observed perinuclear staining for p23 mainly in the cell body of neurons. Double immunofluorescence staining with the cis-Golgi marker, GM130 revealed co-localization of p23 with GM130 in cultured mouse cortical neurons (Fig. 2B). Similarly, in cultured astrocytes p23 staining was found in the Golgi apparatus where it co-localized with GM130 (Fig. 2C). These findings indicate that the main function of p23 in neuronal cells may be similar to that of non-neuronal cells, i.e., the regulation of biosynthetic protein transport. Open in a separate window Fig. 2 Immunofluorescence localization of endogenous p23 in HeLa cells, cortical neurons and astrocytes(A) HeLa cells stably expressing GFP-tagged N-acetylgalactosaminyltransferase-2 were analyzed by immunofluorescence staining with p23 Eptapirone antibody. (B) Primary cortical neurons were stained with p23 antibody and a mAb against Eptapirone the Cav3.1 cis-Golgi marker GM130. Inserts show higher magnification of the dendritic area indicated by the box. p23 co-localizes with GM130 in the cell body and along the dendrites. point to p23 and GM130 co-localization in dendritic Golgi outposts (Horton et al., 2005). (C) Predominant Golgi localization of p23 in cultured astrocytes. Scale bar = 10 m. p23 immunoreactivity in the adult rat brain Having established the specificity of the p23 antibody, we analyzed the localization of p23 in the brain using frozen sections prepared from the adult rat brain. Results showed that p23 immunoreactivity is widely distributed throughout the adult rat brain including the basal forebrain, basal ganglia, cerebral cortex, hippocampus, thalamus, hypothalamus, cerebellum, and brainstem Eptapirone (Fig. 3 and Fig 4). We observed region-specific differences in p23 immunoreactivity, which is evident mostly in neurons and fibers but not in glial cells. The specificity of the p23 immunostaining was further established by using preimmune serum, which failed to show specific staining in any given region of the brain (Fig. 4F). In the following sections, we describe the overall distribution profile of p23 immunoreactivity observed in specific regions of the brain. Open in a separate window Fig. 3 Photomicrographs of coronal.
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