Fig. 1

Endogenous IFNγRα is expressed in neocortical layer 5 neurons of late juvenile and adult rats. a, b Semi-quantitative RT-PCR analysis and Western blots were performed on neocortical tissue of P20 (a, late juvenile) and P60 rats (b, adult). Note that PCR analysis and Western blot were done on the same animal, i.e., neocortical tissue of one hemisphere was used for PCR, while the correspondent neocortex was used for Western blotting. The results shown are representative for three independent experiments, respectively. Left panels, semi-quantitative RT-PCR analysis of ifngr1 mRNA, showing transcriptional gene expression of IFN-γR in the neocortex. The size of the expected fragments corresponds to the expected size (552 base pairs for ifngr1 fragment). Loading control, β-actin cDNA fragments (520 base pairs). Right panels, Western blot of neocortical tissue from the same animal. Detected protein bands range from 45 to 100 kDa (96 kDa predicted by the primary structure of IFNGR1). Similar protein species were previously found to specifically bind IFN-γ [30]. Therefore, we regard the bands to be either full-length or fragmented IFN-γRα that might be post-translationally modified. Alexa 488-labeled β-actin was used as loading control. For estimation of the molecular weight, the commercial weight markers Hyperladder I (for PCR; Bioline) and BlueStar Prestained Protein Marker (for Western blot; Nippon Genetics, Tokyo, Japan) were used. c, d Examples of immunohistochemical co-staining of neocortical tissue in layer 5 with anti-IFNγ-Rα (1:500) and anti-MAP2 (neuronal marker) (1:250) in P20 (c) and P60 (d) rats. Left, single scans for accurate z-localization of Alexa 488-labeled IFNγ receptors α and Alexa 568-labeled MAP2. Right, summations of z-stacks consist of 26 (c) or 52 single sections (d)