1. Date of download: 6/22/2016 Copyright © 2016 American Medical Association. All rights reserved. From: β-Secretase Protein and Activity Are Increased in the Neocortex in Alzheimer Disease Arch Neurol. 2002;59(9):1381-1389. doi:10.1001/archneur.59.9.1381 The β-site APP-cleaving enzyme (BACE) protein enzyme-linked immunosorbent assay (ELISA) (A) and activity assay (B). A, For the protein ELISA, BACE from brain homogenates is captured by the anti-BACE C-terminal antibody MAB5308 and detected by the anti-BACE N-terminal antibody PA1-756, followed by horseradish peroxidase (HRP)–linked anti–rabbit IgG (HRP-α-rb) and activation of the QuantaBlu fluorescent substrate (Pierce, Rockford, Ill) by HRP. B, For the BACE-specific activity assay, BACE is also captured by MAB5308, and the quenched fluorescent substrate is added. Cleavage of the substrate releases the fluorescence, which can be quantitated by fluorimetry. Figure Legend:

2. Date of download: 6/22/2016 Copyright © 2016 American Medical Association. All rights reserved. From: β-Secretase Protein and Activity Are Increased in the Neocortex in Alzheimer Disease Arch Neurol. 2002;59(9):1381-1389. doi:10.1001/archneur.59.9.1381 The β-site APP-cleaving enzyme (BACE) protein enzyme-linked immunosorbent assay (ELISA) and BACE enzymatic activity assay using fluorogenic substrate 1. Absolute fluorescence intensity (abs fl int) is not corrected for background fluorescence; adjusted fluorescence intensity (adj fl int) is corrected. A, Standard curve for BACE protein determination. B, Standard curve for BACE- specific enzymatic activity assay. There was a linear increase in BACE activity (as measured by fluorescence intensity of the cleavage product) with increasing concentration of the brain extract (solid circles). The BACE activity was completely abolished by 3µM peptidergic inhibitor P 10 -P 4′ StatVal (Peptides International, Louisville, Ky) (open circles). C, Linear time-dependent increase in BACE substrate cleavage products (fluorescence intensity adjusted for background). The BACE cleavage products were measured at the indicated time points. D, Dose dependence of BACE peptidergic inhibitor P 10 -P 4′ StatVal on BACE activity. E, The pH dependency of BACE activity in our assay system. The pH optimum was approximately 4.0 to 4.5. F, In a serial dilution of brain extract, BACE activity was correlated with BACE protein. (Error bars are ± SE. Error bars not visible in C, D, and E are contained within the data points.) Figure Legend:

3. Date of download: 6/22/2016 Copyright © 2016 American Medical Association. All rights reserved. From: β-Secretase Protein and Activity Are Increased in the Neocortex in Alzheimer Disease Arch Neurol. 2002;59(9):1381-1389. doi:10.1001/archneur.59.9.1381 The β-site APP-cleaving enzyme (BACE) immunoprecipitation and Western blot analysis. Immunoprecipitation of the brain extracts demonstrates bands at approximately 52 kd and 70 kd. The BACE was captured with anti-BACE C-terminal antibody MAB5308 (lane a) or anti-BACE N-terminal antibody PA1-756 (lane b) and probed with MAB5308. Figure Legend:

4. Date of download: 6/22/2016 Copyright © 2016 American Medical Association. All rights reserved. From: β-Secretase Protein and Activity Are Increased in the Neocortex in Alzheimer Disease Arch Neurol. 2002;59(9):1381-1389. doi:10.1001/archneur.59.9.1381 The β-site APP-cleaving enzyme (BACE) protein and specific activity in brains with Alzheimer disease (AD) and controls and correlation with clinical markers. A, Diagnosis demonstrates a significant main effect on BACE protein by analysis of variance (ANOVA) (P<.001), with significantly increased BACE protein levels in the frontal cortex in brains with AD as determined by a post hoc t test (asterisk indicates P =.004). B, Diagnosis also demonstrates a significant effect on BACE activity by ANOVA (P<.001), with significantly increased BACE activity in the temporal cortex (asterisk indicates P =.007) and frontal cortex (asterisk indicates P =.003) in brains with AD as determined by a post hoc t test. C and D, Because BACE is a predominantly neuronal protein, we normalized BACE measures for synaptic and neuronal loss by determining the ratio of BACE protein to synaptophysin protein (BACE Prt/Syn) (C) and the ratio of BACE activity to synaptophysin protein (BACE Act/Syn) (D). Normalized BACE measures are significantly increased in the temporal and frontal cortices in brains with AD (ANOVA, P<.001; asterisks indicate P =.02 [temporal] and P<.001 [frontal] in C and P =.01 [temporal] and P =.001 [frontal] in D as determined by a post hoc t test.) E, The ratio of BACE activity (Act) to BACE protein (Prt) shows an ANOVA effect by diagnosis (ANOVA, P =.01), reflecting an increased ratio in the temporal cortex (asterisk indicates P =.02 as determined by a post hoc t test). F, The BACE activity increases with duration of illness (P =.008) in the temporal cortex in brains with AD. (Error bars are ± SE.) Figure Legend:

5. Date of download: 6/22/2016 Copyright © 2016 American Medical Association. All rights reserved. From: β-Secretase Protein and Activity Are Increased in the Neocortex in Alzheimer Disease Arch Neurol. 2002;59(9):1381-1389. doi:10.1001/archneur.59.9.1381 Biochemical measures of pathological characteristics and β-site APP-cleaving enzyme (BACE) activity. A, Formic acid–extractable Aβ is markedly increased in brains with AD (analysis of variance [ANOVA], P<.001), especially in the temporal neocortex and frontal neocortex (asterisks indicate P<.001 as determined by a post hoc t test) as well as in the cerebellum (asterisk indicates P =.001). B, Synaptophysin is reduced in the temporal and frontal cortices in brains with AD (ANOVA, P<.001; asterisks indicate P<.001 (temporal) and P =.03 (frontal). C and D, Increasing BACE activity is not significantly correlated with increasing formic acid– extractable Aβ levels in brains with AD. Control brain values are also depicted. (Error bars are ± SE.) Figure Legend:

6. Date of download: 6/22/2016 Copyright © 2016 American Medical Association. All rights reserved. From: β-Secretase Protein and Activity Are Increased in the Neocortex in Alzheimer Disease Arch Neurol. 2002;59(9):1381-1389. doi:10.1001/archneur.59.9.1381 Confocal images of β-site APP-cleaving enzyme (BACE) immunostaining (red) in the brain. A, Temporal cortex from a control case immunostained with Cy3-labeled anti-BACE C-terminal antibody MAB5308 demonstrates neuronal expression of BACE in red. B, Human hippocampus immunostained with Cy3-labeled MAB5308 in red and BODIPY fluorescein–labeled anti–glial fibrillary acidic protein (rabbit polyclonal) in green demonstrates BACE staining in neuronal cell bodies and proximal neurites (red) but not in astrocytes (green). C, Human hippocampus immunostained with Cy3-labeled MAB5308 (mouse IgG1) in red, BODIPY fluorescein– labeled phospho-τ (TG3; mouse IgM) in green, and Cy5-labeled Aβ (rabbit polyclonal R1282) in blue. The BACE is expressed in non–tangle-bearing neurons in red, and tangle-bearing neurons in yellow (arrows, double-stained for BACE and TG3). Scale bars indicate 25 µm. Figure Legend: