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. 2010 Jul 7;30(27):9228–9240. doi: 10.1523/JNEUROSCI.0418-10.2010

Figure 5.

Figure 5.

Increase in amyloidogenic APP processing and insoluble Aβ levels in double mutant reln/app mice. A, Representative Western blots of the SDS-soluble supernatant (SN) derived from hippocampal brain lysates of 9-month-old mice using anti-Aβ (6E10) antibodies. B, Semiquantitative analysis involving densitometry of the immunoreactive β-cleaved C-terminal APP fragments (β-stubs), run in duplicate, corrected for nonspecific background and equal loading using β-actin as control, revealed a significant increase in reln/app compared with app (p = 0.049; n = 4). Soluble Aβ-levels were very low, but clearly detectable in samples of reln/app subjects. C, Representative Western blots using anti-Reelin (G10) antibody recognizing both full-length (FL-Reln) and the N-terminal 180 kDa fragment (N-Reln). The N-terminal-specific APP antibody (22C11) antibody recognized in addition to full-length APP (FL-APP) and the soluble α- or β-secretase-cleaved APP ectodomains (sAPP) short N-terminal fragments, presumably representing N-APP. D, Statistical analysis of the densitometrical measurements of FL-APP and sAPP-immunoreactive fragments revealed a significant difference between genotypes as indicated by the reduced FL-APP/sAPP ratio in reln/app compared with app subjects (p = 0.033; n = 4). In addition, a marked shift toward higher N-APP-immunoreactive fragments in 9-month-old reln/app mice was evident, as demonstrated by the significant reduction in the FL-Reelin/N-APP ratio (G) (p = 0.034; n = 4). E, Representative Western blots using anti-Aβ (6E10) antibodies of SDS-insoluble fractions (pellet) of hippocampal brain lysates, which was resuspended in formic acid (FA). F, Quantitative analysis using ELISA with Aβ40- and Aβ42-specific antibodies revealed a significant increase in Aβ peptides in the insoluble fractions of reln/app compared with app littermates in the hippocampus (Aβ40, p = 0.021; Aβ42, p = 0.046) and neocortex (Aβ40, p = 0.016; Aβ42, p = 0.016; n = 7–8). Values are expressed as fraction of total protein content and given as mean ± SEM. *p < 0.05 **p < 0.01, statistical significance based on Mann–Whitney U test.