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. 2011 Jan 7;286(11):9049–9062. doi: 10.1074/jbc.M110.177519

FIGURE 1.

FIGURE 1.

RCAN1 protein is elevated in brains from AD and DS patients. A, characterization of RCAN1 antibody DCT3. Anti-RCAN1 antibody DCT3 targeting the last 20 amino acids of the human RCAN1 C terminus was raised in a rabbit. Cell lysates from HEK293 cells transfected with plasmid constructs pRCAN1-mycHis or its empty vector pcDNA3.1mycHis(c) were separated on a 12% glycine SDS-polyacrylamide gel. RCAN1 was immunoblotted with DCT3 at a dilution of 1:1000 (lanes 3 and 4). 9E10 antibody targeting the Myc tag fused with RCAN1 was used as positive control (lanes 1 and 2). Lanes 5 and 6 show immunoblots with the preimmune serum drawn from the rabbit before immunization. Lanes 7 and 8 were blotted with DCT3 preincubated with the antigen DCT. B, RCAN1 is enriched in the neuronal layers in human hippocampus and cortex. a, immunohistochemical staining with DCT3 antibody showed that RCAN1 is localized in the granule cell layer in human hippocampus (100×). Higher magnification shows its localization in both cytosol and nucleus in neurons. b, RCAN1 is enriched in pyramidal neuronal layers in cortex (100×). Higher magnification (400×) shows that the characteristic morphology of pyramidal neurons was stained by DCT3 antibody targeting the RCAN1 C terminus. C, representative gels showing Western blots of RCAN1 in tissues from the cortex of DS. Cortical tissues from gestational week 17–21 DS abortuses and their age-matched controls were lysed in 4% SDS radioimmune precipitation assay lysis buffer. 150 μg of total protein were separated on a 12% Tris-glycine SDS-polyacrylamide gel. Rabbit polyclonal anti-RCAN1 antibody DCT3 (1:1000 dilution) was used to detect RCAN1. β-Actin was detected by a monoclonal anti-β-actin antibody (AC15 from Sigma) and was used as loading control. D, quantification of Western blots showed that RCAN1 was increased in DS brains. Values represent mean ± S.E. (error bars) (n = 8). *, p = 0.0372 by Student's t test. E, representative gels showing Western blots of RCAN1 in tissues from the cortex of AD patients. F, quantification of Western blots showed that RCAN1 was markedly increased in AD brains. Values represent mean ± S.E. (n = 10). *, p = 0.0121 by Student's t test. Con, control.

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