51-2700

antibody from Invitrogen Antibodies

Targeting: APP AD1

Provider product page for 51-2700

Western blot

Immunohistochemistry

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Antibody data

Antibody Data
Antigen structure
References [107]
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Validations
Western blot [3]
Immunohistochemistry [1]
Other assay [76]

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Product number: 51-2700 - Provider product page
Provider: Invitrogen Antibodies
Product name: beta Amyloid Polyclonal Antibody (CT695)
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: This antibody can be used to specifically detect the beta-amyloid precursor protein. The antibody reacts with full-length (APP695, 751, 770) and N-terminal truncated forms of beta-APP. The antibody can also be used to detect the C-terminal membrane-anchored fragment of beta-APP that remains after alpha- or beta-secretase cleavage. This antibody does not detect the beta-APP product N-terminal to the gamma-secretase cleavage site.
Antibody clone number: CT695
Concentration: 0.25 mg/mL

APP protein structure - 51-2700 shown in red.

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Experimental details: Figure 3 Confocal images of beta-APP stained tissue, coronal plane, frontal sections . In (A) a low magnification image shows the border between the corpus callosum (lower part) and subcortical white matter. A larger number of beta-APP-positive profiles are visible at the border between the corpus callosum and the subcortical white matter, 24 h after high acceleration trauma. The box in (A) indicates the area that is shown in higher magnification in (B) , in which beta-APP-positive profiles have been indicated by arrows (scale bar = 25 mum).

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Experimental details: Fig. 3 Abundant DAI is readily apparent 6 h following cFPI in the micro pig thalamus. Representative photomicrographs of APP immunofluorescence in the thalamus of animals sustaining sham ( a ) or cFPI ( b ). While sham-injured animals had little to no APP labeling, prevalent APP+ axonal swellings, indicative of DAI, were apparent following injury. c Bar graph depicting the average number of APP labeled axonal swellings/ 0.72 mm 2 of thalamic tissue. Graph depicts mean +- standard error of the mean. * p < 0.05. Scale bar: 50 mum

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Experimental details: Figure 4. Regions with altered DTI metrics exhibit histologic abnormalities consistent with injury. Representative photomicrographs of sham ( A-E ) and 0.65J CHIMERA-injured ( F-J ) optic tract. Sections were stained with silver stain ( A , F ), APP ( B , G ), GFAP ( C , H ), IBA-1 ( D , I ), MBP ( E , J ), and NF ( K , L ). Scale bar = 100 mum. Retraction bulbs and axonal varicosities are prominent in magnified photomicrographs of the 0.65J tissue ( M , N ). Scale bar = 10 mum.

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Experimental details: Figure 3 Confocal images of beta-APP stained tissue, coronal plane, frontal sections . In (A) a low magnification image shows the border between the corpus callosum (lower part) and subcortical white matter. A larger number of beta-APP-positive profiles are visible at the border between the corpus callosum and the subcortical white matter, 24 h after high acceleration trauma. The box in (A) indicates the area that is shown in higher magnification in (B) , in which beta-APP-positive profiles have been indicated by arrows (scale bar = 25 mum).

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Experimental details: Figure 1 AGS are rescued by genetic manipulation of App or mGluR 5 blockade. (A) western blot analyses of AbetaPP levels in Fmr1 KO , WT, App HE T and Fmr1 KO /App HET mice (n = 3 male mice per strain, 1 month old). Statistics: one-way ANOVA p

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Experimental details: Fig. 4 AAD-2004 partially reversed the down-regulated expression of calbindin in the brain of Tg-betaCTF99/B6 mice. (A-F) Photomicrographs showing anti-calbindin-stained hippocampus of non-transgenic control mice (WT; A, B), control Tg-betaCTF99/B6 mice (TG; C, D), and Tg-betaCTF99/B6 mice fed with AAD-2004 (TG+AAD-2004; E, F). Photomicrographs with high magnification of rectangles on the left panels (A, C, E) were shown (B, D, F). Note the anti-calbindin immunoreactivity not only in the granular layer (g) of the dentate gyrus, but also in the pyramidal cells (arrows), in the molecular layer (ml) of the dentate gyrus, and in the stratum oriens (so) and stratum radiatum (sr) of the hippocampus. p, pyramidal layer; hi, hilus. Scale bar, 200 um. (G) Representative Western blot images showing the expression of mouse amyloid precursor protein (mAPP), COX-2, calbindin, transthyretin (TTR), phospho-CREB, and beta-actin in the hippocampus. Total 5-8 animals were analyzed for each group.

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Experimental details: Figure 8 Axonal injury as indicated by immunohistochemical staining for neurofilament heavy-chain (""NF-H"") OR beta-amyloid precursor protein (APP). Exemplar coronal section (x6 magnification) 1.3 mm caudal of bregma analysed for staining for NF-H and for beta-APP is shown in ( A ). The boxes indicate the sub-ventricular zone (Box 1; SVZ) or the corpus callosum (Box 2; CC) shown in greater detail (x20 magnification) in the next four rows of panels. ( B, C ) Staining in a TBI animal 24 hrs post-TBI for NF-H (B1, C1) or beta-APP (B2, C2) in the SVZ (row B) or the CC (row C). ( D, E ) As for ( B, C ) for equivalent brain regions, from a Sham control animal. Scale bar indicates 50 um.

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Experimental details: Figure 4 BMS-869780 had minimal effect on APP-CTF accumulation in vitro . H4-APPsw cell cultures were treated overnight with the indicated concentrations of BMS-869780, BMS-299897 or vehicle (0.1% DMSO). (a) Cells were harvested and analyzed by western blotting for APP-CTF alpha , APP-CTF beta , and GAPDH. Lane 1; culture treated with vehicle 0.1% DMSO. Lanes 2-5; cultures treated with BMS-869780 at 100 nM, 300 nM, 1000 nM, or 3000 nM, respectively. Lanes 6-8; cultures treated with BMS-299897 at 30 nM, 100 nM, or 300 nM, respectively. (b) Levels of A beta 1-42 (red; left Y -axis), A beta 1-40 (green; right Y -axis), and A beta 1- x (grey; right Y -axis) were quantified.

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Experimental details: Fig 6 IDN5706 inhibits proteolytic processing of APP to CTFs, and production of Abeta species. (A) Schematic representation of APP and carboxy-terminal fragments (CTFs) indicating their topological domains and the position of the proteolytic cleavage sites by alpha, beta and gamma secretases. The cytosolic region, recognized by the anti-tail antibody, and the gamma-secretase inhibitor, DAPT, are indicated. (B) H4 cells were left untreated (lane 1) or treated for 16 h either with 250 muM IDN5706 (lane 2), 1 muM DAPT (lane 3), or with a combination of 250 muM IDN5706 and 1 muM DAPT (lane 4). Cell extracts were subjected to Western blot analysis using the anti-tail antibody to the cytosolic C-terminal region of APP. Western blotting with antibody to beta-actin was used as loading control. mAPP, mature APP; iAPP, immature APP. The position of molecular mass markers is indicated on the left. (C-D) CHO 7AP2 cells were cultured in DMEM containing low glucose and without fetal bovine serum, in the absence or presence of 250 muM IDN5706 for 16 h. The amount of Abeta40 and Abeta42 peptides in the culture medium was analyzed by ELISA. (E) Ratio of the amount of Abeta42 and Abeta40 peptides as an indicator of toxicity. (C-E) Values are presented as the mean +- SD of three independent experiments. *, P < 0,05 and **, P < 0,005.

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Experimental details: Fig 7 IDN5706 disrupts glycosylation of APP. (A) H4 cells stably expressing an amyloidogenic version of APP tagged to GFP (APP-GFP) were treated with 250 muM IDN5706 for the indicated periods of time. Cell extracts were subjected to Western blot analysis with an antibody to GFP. Western blotting with antibody to beta-actin was used as loading control. (B) H4 cells stably expressing APP-GFP, were left untreated (lanes 1-3) or treated for 16 h with 250 muM IDN5706 (lanes 4-8). Cell extracts were subjected to immunoprecipitation with a an antibody to GFP, followed by denaturation and digestion with the indicated glycosidases for 1 h at 37degC. Immunoprecipitated proteins were subjected to Western blot analysis with anti-GFP-HRP. (A-B) The position of molecular mass markers is indicated on the left. mAPP, mature APP; iAPP, immature APP.

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Experimental details: Fig 9 Accumulated immature APP in response to IDN5706 is degraded by Atg5-dependent autophagy. (A) H4 cells stably expressing an amyloidogenic version of APP tagged to GFP, and stably expressing either luciferase shRNA (control; shLuc) or Atg5 shRNA (shAtg5), were treated with 250 muM IDN5706 for the indicated periods of time, or (C) were left untreated (lanes 1 and 6) or treated with 250 muM IDN5706 (lanes 2-5 and lanes 7-10) for 12 h, followed by cycloheximide-chase with 150 mug/ml cycloheximide and 40 mug/ml chloramphenicol for 1-3 h in the presence of 250 muM IDN5706 (lanes 2-5 and lanes 7-10). Equivalent amounts of cell extracts were subjected to SDS-PAGE in 7.5% acrylamide gels, followed by Western blotting with an antibody to GFP. Western blotting with antibody to beta-actin was used as loading control. mAPP, mature APP; iAPP, immature APP. The position of molecular mass markers is indicated on the left. (B and D) Densitometric quantification of the levels of iAPP shown either in A or C. The values depicted in the graphs represent the mean +- SD of three independent experiments. *, P < 0.05; **, P < 0.01.

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Experimental details: Fig. 2 Axonal injury is observed in various regions throughout the micro pig brain following cFPI. Representative photomicrographs of APP immunohistochemistry in regions of the micro pig brain that demonstrated DAI in animals sustaining cFPI. Images in the middle panel ( b , f , i , l , o , r ) are magnified regions indicated in the images of the left panel ( a , e , h , k , n , q ) and images in the right panel ( c , g , j , m , p , s ) are magnified regions indicated in the middle panel ( b , f , i , l , o , r ), respectively. Note that DAI within the thalamus and tectum was diffusely distributed throughout the domain, while DAI within the other regions was more localized. Also note that while not common, APP+ proximal axonal swellings in continuity with the neuronal soma ( d ) were observed in the thalamus. Scale bar in q : 200 mum; r and s : 100 mum; d : 50 mum

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Experimental details: Fig. 3 Abundant DAI is readily apparent 6 h following cFPI in the micro pig thalamus. Representative photomicrographs of APP immunofluorescence in the thalamus of animals sustaining sham ( a ) or cFPI ( b ). While sham-injured animals had little to no APP labeling, prevalent APP+ axonal swellings, indicative of DAI, were apparent following injury. c Bar graph depicting the average number of APP labeled axonal swellings/ 0.72 mm 2 of thalamic tissue. Graph depicts mean +- standard error of the mean. * p < 0.05. Scale bar: 50 mum

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Experimental details: Fig. 6 Microglia activation occurs in thalamic sectors sustaining acute DAI 6 h following mTBI. Representative confocal micrographs of APP ( red ; a and d ) and Iba-1 ( green ; b and e ), with overlays in c and f , in the thalamus of the same injured animal. Interestingly, areas lacking axonal injury ( a - c ) appear to contain inactive ramified microglia ( arrows ), whereas thalamic sites exhibiting DAI, indicated by accumulation of APP in axonal swellings ( d - f ), also appear to contain the majority of morphologically activated Iba-1+ microglia ( arrow heads ). Scale bar: 20 mum

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Experimental details: Fig. 7 Microglia processes appear to preferentially contact TBI-induced proximal axonal swellings. Representative 3D reconstructions of MBP+ myelinated axons ( red ) or APP+ axonal swellings ( green ) and Iba-1+ microglia ( white ) in sham-injured ( a ) or central fluid percussion injured ( b ) thalami. c Bar graph depicting the average number of Iba-1+ microglial processes contacting either MBP+ myelinated fibers in the sham animals or APP+ axonal swellings in injured animals. Graph depicts the mean +- standard error of the mean. * p < 0.05. Scale bar: 5 mum

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Experimental details: Fig 2 Axonal transport effects, APP labeling with losartan-treated vs water control. Immunolabeling for APP in the optic nerve head of mice treated with oral losartan (A and C) and water alone (B and D) shows axonal transport obstruction 3 days after IOP elevation. The examples show 4 of the 5 grade levels for APP accumulation (level 1, not illustrated, had no label, indicating that there was no accumulation and no background or non-specific labeling). The grades of losartan-treated eyes ranged from 2 to 4 (A and C; mean = 2.6 (sd 0.8), while water-treated eyes ranged from 3 to 5 (B and D; mean = 4.0 (sd 0.9); difference from losartan, p = 0.007, multivariable regression adjusted for IOP exposure). Bar equals 50 um.

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Experimental details: Fig. 3 Effects of CCR2 signaling on axonal damage after TBI. Axonal pathology was evaluated at 3 dpi in control heterozygous and Ccr2 RFP / RFP mice by APP staining. a The injury induces APP accumulation in axons of the ipsilateral side to the injury, particularly in the corpus callosum. Ccr2 deletion decreases APP accumulation in axons. Scale bar , 500 mum. b Higher magnification images of the indicated regions ( boxes ) show that APP immunoreactivity aligns with axonal tracks in the corpus callosum (CC). Scale bar , 100 mum. c Quantification of axonal pathology as percent area of the corpus callosum with APP immunoreactivity above threshold. Statistics: two-way ANOVA and Tukey's post hoc test. Comparisons between groups are shown with horizontal lines ; vertical line in figure legend indicates main effect of genotype. * p < 0.05; ** p < 0.01

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Experimental details: Figure 5 BMS-869780 modulated A beta but did not cause accumulation of beta CTF or alpha CTF in rat brain. Rats were given oral doses of BMS-869780, and levels of brain A beta , beta CTF, and alpha CTF were determined 24 hours later. For comparison, BMS-698861 was dosed in a separate experiment and samples were taken 5 hours later. (a) Brain levels of A beta 1-42 (red), A beta 1-40 (green), A beta 1-38 (blue), and A beta 1-37 (purple) are shown as bars stacked upon one another. The total height of each bar therefore represents the sum of the four peptides. (b) A beta 1-42 (red--left Y axis) and A beta 1- x (grey--right Y axis). The same results for A beta 1-42 are plotted in both (a) and (b). (c) Rat brain beta CTF was detected by western blotting of immunoprecipitates from samples of the same rat brains used for A beta determinations. V, vehicle groups; results from rats dosed with 1.9, 22, 100, and 235 mg/kg of BMS-869780 and 10 mg/kg BMS-698861 (GSI) are indicated. (d) Western blots of immunoprecipitated alpha CTF from the same rat brain samples. (e) and (f) quantification of western blots shown in (c) and (d), respectively, expressed relative to percent of average level of CTF in vehicle-treated rats. Actual doses of BMS-869780 were determined by analysis of concentrations in left-over dosing solutions.

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Experimental details: FIGURE 5 Amyloid precursor protein expression in FXTAS patients and mouse models. (A) Frontal cortex lysates from control ( n = 10) and FXTAS patients ( n = 3) probed with a C-terminal alphaAPP antibody which detects the three primary isoforms of full-length APP (100-125 kDa). (B) Cerebellar lysates from the same individuals as in (A) . (C) Normalized APP expression relative to tubulin expressed as a percent of controls, performed in technical triplicate. (D) Twelve-month-old CGG KI ( n = 4) and WT littermate control ( n = 3) cortex and subcortical lysates probed with alphaAPP. (E) Cerebellar lysates from the same animals as in (D) . (F) Normalized APP expression in CGG KI mice expressed as a percent of WT controls, performed in technical triplicate. * P < 0.05 Student's t-test.

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Experimental details: Figure 3 (a) Normal looking axons in the white matter tracts of cervical spinal cord of a 24 h survival sham animal (b) axons with prominent swellings (arrowheads) and swollen axons with vacuolations (arrows) in the white matter tracts of cervical spinal cord at 6 h (c and d) prominent disruptions of axonal membranes (arrowheads) at 6 h and 24 h respectively. These disruptions in the form of wide spaces on the margins or projections with ragged edges could be seen in various large caliber axons (e) axonal injury in the form of vacuolations in the axonal core at 24 h (f) a beta amyloid precursor protein reactive swollen axon in the cervical spinal cord white matter tract at 6 h post blast period (g) beta amyloid precursor protein reactive punctate axons 24 h post blast

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Experimental details: Figure 2 PSMD14 is validated as a regulator of the endogenous APP levels. ( A ) Protein extracts of parental H4 cells either untransfected (Mock), transfected with NT siRNA, or transfected with four different PSMD14 siRNA sequences for 72 h were analyzed by western blot. Polyclonal antibodies to endogenous APP (CT695) and to Ub (that recognizes all types of Ub conjugates), and monoclonal antibodies to PSMD14 (clone D18C7) and to beta-actin (clone BA3R), were tested. The position of molecular mass markers are indicated on the left. Densitometric quantification of the levels of endogenous APP ( B ) and PSMD14 ( C ) in H4 cells transfected with PSMD14 siRNA#1, compared to untransfected cells (Mock). Statistical significance was determined by Student's t-test. Bars represent the mean +- SD of biological replicates (APP n =5; PSMD14 n = 4). ** p < 0.01 and *** p < 0.001. ( D ) mRNA levels of psmd14 and ( E ) mRNA levels of app were measured using RT-qPCR from parental H4 cells transfected for 72 h. All data were normalized for TATA binding protein expression in either untransfected cells (Mock), cells transfected with NT siRNA or cells transfected with four different PSMD14 siRNAs duplexes. Statistical significance was determined by One-Way ANOVA, followed by Tukey's test. Bars represent the mean +- SD of biological replicates ( psmd14 n = 3; app n = 3). Different letters above the mean bars apply to significant differences between groups p < 0.01.

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Experimental details: Figure 3 Acute inhibition of PSMD14 by Capzimin CZM shows a similar phenotype as that of PSMD14 KD on the levels of APP and high molecular weight Ub conjugates. ( A ) Schematic diagram of the molecular targets of Capzimin and MG132 in the 19S RP and 20S catalytic core of the proteasome, respectively. ( B ) Parental H4 cells were treated either with vehicle (DMSO; Control), or increasing doses of CZM for 4 h, or MG132 for 6 h. Protein extracts were analyzed by western blot with a polyclonal antibody to endogenous APP. Monoclonal antibody to beta-actin (clone BA3R) was used as a loading control. The position of molecular mass markers is indicated on the left. ( C ) Densitometric quantification of APP protein levels as shown in ( D ). Statistical significance was determined by one-way ANOVA, followed by Tukey's test. Bars represent the mean +- SD of biological replicates ( n = 4). Different letters above the mean bars apply to significant differences between groups p < 0.05. ( D ) Parental H4 cells were treated as in ( B ), and the protein extracts were analyzed by western blot with a polyclonal antibody to Ub that recognizes all types of Ub conjugate. Monoclonal antibody to beta-actin (clone BA3R) was used as a loading control. The position of molecular mass markers is indicated on the left. ( E ) Immunofluorescence microscopy images of the cellular localization of Ub in parental H4 cells treated with either the vehicle (DMSO; Control), CZM for 4 h or MG132 for 6 h. Cells were

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Experimental details: Figure 4 Acute inhibition of PSMD14 by CZM triggers the accumulation of APP in a swollen Golgi apparatus. Immunofluorescence analysis of endogenous APP in H4 parental cells treated either with the vehicle (DMSO; Control) ( A - C ) or CZM ( D - F ) for 4 h. Cells were fixed, permeabilized, and double stained with a rabbit polyclonal antibody to APP (CT695) ( A , D ) and a mouse monoclonal antibody to GM130 (clone35/GM130) ( B , E ), followed by Alexa-594-conjugated donkey anti-Rabbit IgG and Alexa-488-conjugated donkey anti-Mouse IgG. Merging of the images generated the third picture ( C , F ). Scale bar, 10 mm. ( G ) Quantitative analysis of the mean of total fluorescence intensity of APP upon treatment with CZM, in comparison to control cells. The statistical significance was determined by Student's t-test. Bars represent the mean +- SD of the fluorescent signal per cell area ( n = 43 cells). *** p < 0.001. ( H ) Quantitative analysis of the fraction of APP colocalizing with GM130 under CZM treatment and compared to control cells. Statistical significance was determined by Student's t-test. Bars represent the mean +- SD of the fluorescent signal per cell area ( n = 43 cells). *** p < 0.001. ( I ) Quantitative analysis of the cell area. Statistical significance was determined by Student's t-test. Bars represent the mean +- SD of the cell area ( n = 43 cells) ** p < 0.001. ( J ) Immunofluorescence microscopy analysis of GM130 in parental H4 cells treated either with the vehicl

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Experimental details: Figure 3 Amyloidogenic precursor protein (APP) processing analysis after single and repeated instillations of BB and DEP. Representative immunoblotting images of amyloid precursor protein (APP), phosphorylated APP on threonine 668 (p-APP Thr668 ), and beta-secretase 1 (BACE1) analysis in mice after single ( A ) and repeated ( E ) instillations with 50 µg of BB or DEP/100 uL 0.9% NaCl. Histograms display p-APP Thr668 /APP, APP, and BACE1 protein levels in mice after single ( B - D ) and repeated ( F - H ) instillations with BB and DEP, with respect to sham. Proteins are normalized to corresponding total proteins revealed by Ponceau in each lane ( Figure S3, Supplementary Materials ), and the data are expressed as means +- SEM ( n = 6). Statistical differences were tested accordingly by one-way ANOVA followed by Tukey post hoc comparison. ** p < 0.01 vs. sham mice; **** p < 0.0001 vs. sham mice; SS p < 0.05 vs. BB-treated mice.

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Experimental details: Figure 3 Representative micrographs of amyloid precursor protein (APP) immunofluorescence in the thalamus of pigs sustaining central fluid percussion injury (cFPI) followed by (A) vehicle or (B) levetiracetam (LEV) treatment. (C) Bar graph depicting the average number of APP-labeled axonal swelling/unit area of thalamic tissue at 6 h post-cFPI (vehicle, n = 7 pigs; LEV, n = 7 pigs). The number of axonal swellings at 6 h was not significantly different with LEV treatment vs. vehicle. Scale = 100 um.

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Experimental details: Axonal injury indicated by post-injury accumulation of APP . (A-C) Example of APP immunostaining from the external capsule. (A and J) No APP accumulation was observed in sham-injured controls. (B and C) Following TBI , APP accumulation was observed, showing both the classical varicosities (open arrows) and axonal bulbs (white arrows) in both TBI models. (D-I) Schematic overview of post-injury accumulation of APP in the two TBI models from 2 to 21 days post-injury. Each asterisk indicates a region of APP -positive axonal profile in the evaluated white matter regions that was not observed in sham-injured controls. Note the decreased APP immunostaining at the late time points in both TBI models. CC , corpus callosum; EC , external capsule; F, fimbriae; PID , post-injury day.

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Experimental details: Fig. 10 Pharmacological inhibition of FT reverses the hyperactivation of Ras-Rheb-mTORC1 signaling in SH-SY5Y-APP695 cells. A Immunoblot analysis of FL-APP and downstream targets of Ras-Rheb-mTORC1 signaling, including ERK and ribosomal protein S6, in the lysates of SH-SY5Y-APP695 and mock-transfected control cells. B Densitometric analysis of immunoblots normalized by the amount of GAPDH or total proteins for phosphorylated proteins (P-ERK, P-Akt, P-S6) with the levels in the control group set as 100%. n = 6 replicates from 2 independent experiments. C Immunoblot analysis of farnesylation status of H-Ras and Rheb, and D phosphorylation status of downstream targets of Ras and Rheb in the lysates of SH-SY5Y-APP695 cells after treated with vehicle (DMSO) or 1 muM tipifarnib for 48 h. E Densitometric analysis of immunoblots of P-ERK, P-S6K, and P-S6 normalized by the amount of total ERK, S6K, and S6 with the levels in the control group set as 100%. n = 6 replicates from 2 independent experiments. Student's t-test, 2-tailed; * p < 0.05; *** p < 0.001; **** p < 0.0001

Supportive validation

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Experimental details: Fig. 7 Reduced APP proteolytic processing in APP/PS1/FTnKO mice. A , B Immunoblots and densitometric analysis of full-length APP (FL-APP), and APP cleavage products, carboxyl-terminal fragments (CTFalpha and CTFbeta), amino-terminal fragments of APP (sAPPalpha and sAPPbeta), and beta-secretase 1 (BACE1). Protein levels were normalized by the amount of tubulin with the levels in the APP/PS1 control group set as 100%. C , D Immunoblots and densitometric analysis of proteins involved in Abeta clearance pathways including APOE, LRP1, IDE, and neprilysin. Protein levels were normalized by the amount of tubulin or GAPDH, and the levels in the control group set as 100%. n = 8/genotype; Student's t-test, 2-tailed; # p = 0.08; * p < 0.05

Supportive validation

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Experimental details: Figure 1 ( A ) Effects of aging on protein expression of APP in the aortas of wild-type (WT) littermates and eAPP -/- mice (n=10 per group). ( B ) Effects of aging on ex-vivo sAPPalpha secretion from wild-type (WT) littermates and eAPP -/- mice aortas. The supernatants were collected and analyzed for sAPPalpha levels. Results were normalized against tissue protein levels (n=12 per group for young WT littermates and eAPP -/- mice and n=15 per group for aged WT littermates and eAPP -/- mice). ( C ) Effects of aging on protein expression of ADAM10 in the aortas of wild-type (WT) littermates and eAPP -/- mice (n=6 per group). ( D ) Effects of aging on protein expression of BACE1 in the aortas of wild-type (WT) littermates and eAPP -/- mice (n=6 per group). Western blot results are the relative densitometry compared with beta-actin protein. All results are representing box plots with whiskers showing the median, 25 th to 75 th percentiles, and min-max range. * P

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Experimental details: Figure 9 Effects of ex-vivo treatment for 24 hours with cytokines cocktail on protein expressions of APP ( A ) and eNOS ( B ) in the aortas of young wild-type (WT) littermates and eAPP -/- mice. Western blot results are the relative densitometry compared with beta-actin protein (n=9 per group). All results are representing box plots with whiskers showing the median, 25 th to 75 th percentiles, and min-max range. * P

Supportive validation

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Experimental details: Figure 3 Buprenorphine did not alter acute diffuse axonal injury in either the thalamus or cortex. Representative photomicrographs of ( A , C ) saline or ( B , D ) buprenorphine treated Sprague Dawley rats 1d following cFPI. Tissue samples of ( A , B ) somatosensory neocortex and ( C , D ) hemithalamus were labeled immunohistochemically with a primary antibody against amyloid precursor protein (APP) and a secondary biotinylated antibody followed by DAB reaction. Swellings were visualized and quantified to assess axonal injury in each brain region at 1d following cFPI and either saline or buprenorphine treatment. ( E , F ) Corresponding bar graphs depicting the number of swellings per region of interest in the ( E ) cortex and ( F ) thalamus. The number of swellings was consistent between saline and buprenorphine treated animals, indicating that buprenorphine does not affect axonal injury in observed regions following TBI. Figure was compiled using Adobe Photoshop CS., version 22.0 (2020), San Diego, CA. n = 6 rats/group; Mean +- SEM. scale = 50 mum.

Supportive validation

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Experimental details: Fig. 3 Sarm1 knockout reduces corpus callosum atrophy, myelin loss, and axon damage at 10 weeks post-TBI. a - d Representative images from CC coronal sections of Sarm1 WT and Sarm1 KO mice after sham or TBI procedures. Myelin is detected with immunolabeling for MOG (red). DAPI nuclear stain is shown in blue. Insets show axonal swellings with beta-APP immunoreactivity (examples indicated by arrows). CC borders are indicated by dashed lines. e Sarm1 knockout attenuates CC atrophy, which is quantified based on the CC width. f TBI results in significant myelin loss as detected by reduced MOG immunoreactivity. Myelin loss after TBI is significantly reduced in Sarm1 KO mice compared to Sarm1 WT mice. g Axon damage in TBI mice was significantly reduced in Sarm1 KO mice compared to Sarm1 WT mice. MOG quantification included Sarm1 WT: n = 7 sham, n = 11 TBI; Sarm1 KO: n = 6 sham, n = 11 TBI. beta-APP quantification included Sarm1 WT: n = 6 TBI; Sarm1 KO: n = 9 TBI. ns = not significant. Further statistical details are provided in Additional File 1 : Table S3. Scale bars a - d = 100 um, insets b , d = 25 um

Supportive validation

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Experimental details: Fig. 2 Hyperoxygenation treatment increased the expression of MMP-2, MMP-9, and tPA in the hippocampus of Tg-APP/PS1 mice. (A) Real-time PCR data showing transcript levels of Bace1 , Bace2 , Psen1 , Psen2 , Adam10 , and Apoe genes in the hippocampus of wildtype control (WT-CON), wildtype mice exposed to hyperoxygenation (WT-HO 2 ), Tg-APP/PS1 control mice (Tg-CON), and Tg-APP/PS1 mice exposed to hyperoxygenation (Tg-HO 2 ). n=6-7 animals/group, 4 PCR repeats. (C) Western blot data showing expression levels of APP, BACE1, PSEN1, and ApoE in the hippocampus of WT-CON, WT-HO 2 , Tg-CON, and Tg-HO 2 mice. n=6~8 animals/group, 4~11 repeats. (D) Real-time PCR data showing transcript levels of Mmp-2 , Mmp-9 , tPA , Ide , Nep , and Lrp1 genes in the hippocampus of WT-CON, WT-HO 2 , Tg-CON, and Tg-HO 2 mice. n=6~7 animals, 4 data points. (E) Western blot data showing expression levels of MMP-2, MMP-9, tPA, uPA, and Ide genes in the hippocampus of WT-CON, WT-HO 2 , Tg-CON, and Tg-HO 2 mice. Tissue samples were prepared at the time point of Figure 1A . n=6~8 animals/group, 4~10 data points. Data are presented as mean+-SEM. * , **difference between indicated groups. *p

Supportive validation

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Experimental details: Figure 3 MOAB-2 detection of intraneuronal Abeta but not intraneuronal APP in 5xFAD brain tissue . Immunofluorescent detection of Abeta and APP in the cortex of: (A) 1-month old 5xFAD mice using: MOAB-2, Abeta42- or Abeta40-specific antibodies; (B) 1-month old 5xFAD mice with MOAB-2 and antibodies against the C-terminus (Cter) (CT695) or N-terminus (Nter) (2211) of APP; and (C) 4-month old 5xFAD/BACE -/- with 6E10 or MOAB-2, plus antibodies CT695 and 22C11. (Coronal sections, representative confocal images at 100x). Antibody concentrations: MOAB-2 (1:1000), anti-Abeta40, (1:1700), anti-Abeta42 (1:200), 22C11 (1:40), CT695 (1:500), 6E10 (1:1000).

Supportive validation

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Experimental details: Figure 2 Curcumin analog C1 activates transcription factor EB (TFEB), degrades beta-amyloid precursor protein (APP) and beta-amyloid peptides (Abeta), and prevents synaptic and cognitive failures in 5xFAD mice. (a, b) Western blots showed the levels of full-length APP (Fl-APP), CTF-beta/alpha, Abeta, LAMP1, CTSD, LC3B, cytosolic and nuclear TFEB (cTFEB and nTFEB) with their respective loading controls in the brain lysates of male mice (Another batch of blots for male mice and all blots for female mice were shown in Figure S2 B). (c, d) All the values from male ( n = 6) and female ( n = 6) mice were quantified as average +- SEM . * p < .05, ** p < .01, and *** p < .001 vs. Veh. group, respectively, analyzed by one-way ANOVA. The interaction between sex and drug effects was analyzed by two-way ANOVA, and the P values were summarized in Table S1 . (e, f) Immunohistochemistry analysis of Abeta load. (e) Representative images show Abeta labeled with biotinylated 4G8 antibody. (f) Data are quantified as mean +- SEM in male ( n = 6) and female ( n = 6) mice. ** p < .01 and *** p < .001 vs. Veh. group, respectively. (g) ELISA assay of Abeta42 and Abeta40 in male ( n = 6) and female ( n = 6) mice. Whole-brain samples were separated into Triton soluble and formic acid (FA) fractions. Data were presented as mean +- SEM . # p < .05, ## p < .01, and ### p < .001 vs. Veh. group (Abeta42); * p < .05, ** p < .01, and *** p < .001 vs. Veh. group (Abeta40). The interaction between sex and drug

Supportive validation

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Experimental details: Figure 4 Curcumin analog C1 activates transcription factor EB (TFEB) and attenuates both beta-amyloid precursor protein (APP) and Tau pathology in 3xTg mice. (a, b) Representative blots showed the levels of Fl-APP, CTF-beta/alpha, phosphorylated Tau (PHF-1, AT-8), total Tau, LAMP1, CTSD, cytosolic and nuclear TFEB (cTFEB and nTFEB), and LC3B with their respective loading controls in the hippocampus of female mice (Another batch of blots were shown in Figure S3 A). (c, d) Data are quantified as mean +- SEM ( n = 6). * p < .05, ** p < .01, *** p < .001 vs. Veh. treatment analyzed by one-way ANOVA. (e, f) Immunohistochemistry analysis of beta-amyloid peptides (Abeta) load. (e) Representative images show the Abeta labeled with biotinylated 4G8 antibody. (f) Data are quantified as mean +- SEM ( n = 10). * p < .05 vs. Veh. treatment analyzed by one-way ANOVA. (g, h) Immunohistochemistry analysis of p-Tau (AT8). (g) Representative images show AT8 staining in the posterior hippocampus. (h) Data are quantified as mean +- SEM ( n = 7-12). * p < .05, ** p < .01 vs. Veh. treatment analyzed by one-way ANOVA. CTSD, cathepsin D; LAMP, lysosomal-associated membrane protein 1

Supportive validation

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Experimental details: Fig. 1 Mechanical tensile strain of 30% induces axonal injury in organotypic cerebellar slice culture. a Ex vivo culture and 30% strain model using organotypic slice cultures of cerebellum. b Double immunostaining of organotypic cerebellar slices for APP in red and SMI-312 (pan-axonal neurofilament marker) in green. Images represent control (Ctrl) and stretched (Stretch) cultures. The response of slices was evaluated at time 0 h post-stretch. Scale bar, 50 mum. c High magnification of the white square in the stretch panel from b . Scale bar, 20 mum. d Quantitative analysis of the number of APP aggregates per slice. Results represent the mean +- SEM ( n = 3). * p < 0.05. APP amyloid precursor protein, DIV days in vitro

Supportive validation

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Experimental details: Figure 1 Central fluid percussion injury does not result in focal brain damage, but does precipitate diffuse axonal injury in the thalamus of both rats and micro pigs 1 day following injury. Representative photographs of the gross (A,C) micro pig and (B,D) rat brain 1 day following cFPI. The top panels are dorsal and ventral views of the entire brain 1 day post-injury while panels (C) and (D) represent 5 mm thick coronal sections at the level of the rostral thalamus in the (C) micro pig and (D) rat. The boxes indicate the regions of analysis of microglia process convergence in the thalamus of both species. Diffuse cFPI also did not result in cell damage/death. Hematoxylin and eosin staining reveled no square wave cellular damage in the thalamus of either the (E) micro pig or (F) rat thalamus at 1 day following cFPI. Amyloid precursor protein (APP) immunohistochemistry in the (G) micro pig and (H) rat thalamus, however, demonstrated diffuse axonal injury (arrows) 1 day following cFPI. Insets (e-h) depict higher magnification images of panels (E-H) , respectively. Note that the cFPI model employed did not result in contusion, hematoma formation or square wave cell damage/death, however did demonstrate diffusely distributed axonal injury, consistent with diffuse injury. Scale bar A-D : 1 mm; E-H : 100 um; e-h: 50 um.

Supportive validation

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Experimental details: Figure 3 APP expression in the corpus callosum. Fluorescence microscopy photographs of the corpus callosum (coronal sections) at around 1mm from bregma (top row) and around -3 mm from bregma (bottom row). ( A , F ) The highlighted area refers to the area represented in the photographs on the same row. ( C - E ) Following injury, axonal bulbs localize mostly at the borders between the corpus callosum and grey matter. Insert show details of the CC-lateral ventricle border area. ( H - J , M ) Moving occipitally, APP expression is comparatively less. ( B , G ) Sham animals do not show APP accumulation in the axonal terminals at the regions of interest. ( K - M ) Scatter plots showing the amount of APP+ profiles in each ROI (y-axis represents the number of APP+ profiles; x-axis represents each observation). APP expression is maximal at 24 h and decreases thereafter, with p

Supportive validation

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Experimental details: Figure 4 Olig2 expression in the corpus callosum. ( A , F ) The highlighted area refers to the area represented in the photographs on the same row. ( B - E ) Olig2 expression in the corpus callosum, at its interface with the lateral ventricle at around 1 mm from bregma. ( G - J ) Olig2 expression in the corpus callosum at around -3 mm from bregma. Scale bar = 200 um.

Supportive validation

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Experimental details: Figure 12 HSP70/ATF3/APP expression reflecting neuronal stress. ( A , F ) The highlighted area refers to the area represented in each row. ( B - E ) HSP70 (red) and APP (green) double staining in the dorsal cortex. While APP expression remains elevated through 7 d, HSP70 expression is comparatively decreased at 7 d. Few neurons are HSP70+/APP+. ( G - J ) ATF3 (red) and APP (green) double staining in the habenular nuclei. ATF3 follows the same expression pattern as HSP70, with decreased marker uptake at 7 d. In all cases, sham animals do not express any of the tested markers, with only the baseline marker uptake visible. Scale bar = 150 um (Scale bar = 100 um in image ( F )).

Supportive validation

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Experimental details: Figure 13 HSP70/APP double-staining in 24 h trauma-exposed animals. ( A , C ) The highlighted area refers to the area represented in the adjacent microphotograph. ( B ) HSP70 and APP expression in the cingulate cortex. Few neurons are positive for both markers. ( D ) HSP70 and APP expression in the caudoputamen, close to its interface with the corpus callosum. Scale bar = 200 um.