13-0200

antibody from Invitrogen Antibodies

Targeting: APP AD1

Provider product page for 13-0200

Western blot

ELISA

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [57]
Comments [0]
Validations
Western blot [1]
Immunohistochemistry [1]
Other assay [42]

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Product number: 13-0200 - Provider product page
Provider: Invitrogen Antibodies
Product name: Amyloid Precursor Protein Monoclonal Antibody (LN27)
Antibody type: Monoclonal
Antigen: Other
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: LN27
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

APP protein structure - 13-0200 shown in red.

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Experimental details: Figure 1 SIV infection induced encephalitis, increased CNS CD68 expression, and axonal accumulation of APP. SIV-infected pig-tailed macaques that developed encephalitis had multifocal perivascular accumulations of infiltrating macrophages and multinucleate giant cells (A, arrows denote giant cells, * denotes blood vessel lumen, hematoxylin and eosin stain, bar = 50 uM), increased CNS CD68 expression reflecting activation of macrophages and microglia in subcortical white matter shown by immunohistochemical staining with an anti-CD68 antibody (B, bar = 50 uM), and accumulation of APP in axons (arrows) in the corpus callosum (C, bar = 100 uM).

Supportive validation

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Experimental details: Figure 1 Amyloid pathology and glial inflammation in ARTE10 mouse brain. A , Double-immunofluorescent colocalization of the transgenic human APP ( red , human-specific antibody against the N-terminal ectodomain) with the neuron-specific somato-dendritic marker MAP2 ( green ) in the subiculum and the CA1 region of the hippocampus. The transgene is amply expressed in most (>80%) neurons ( yellow merge colour), whereas non-neuronal cells show no signals beyond background. B , Congo-red stained plaque core ( red ) in the frontal cortex of a hemizygous ARTE10 mouse at 5 months of age. The green bi-refringence in polarized light (inset) is diagnostic of amyloid. C, D , Thioflavin-S positive plaque cores ( green ), each encircled by a sphere (corona) of dilated, strongly ubiquitin-positive dystrophic neurites ( red ) in the subiculum of a homozygous ARTE10 mouse at 3 months of age (D) and in the frontal cortex of a 19 months old hemizygous mouse (C), the latter with concomitant amyloid angiopathy (arrow). E, F , AT8-positive hyperphosphorylated tau ( red ) in dystrophic neurites adjacent to plaque cores ( green : Abeta40 and Abeta42) in homozygous ARTE10 mice at 12 (E) and 8 (F) months of age. G, H , Triple-immunofluorescent demonstration of plaque-associated mixed glial inflammation: Activated microglia ( green , AIF1/Iba1) and reactive astroglia ( magenta , GFAP) colonizing the periphery of amyloid cores ( red : Abeta/6E10) in the subicula of a homozygous 5 months (G) and a hemizy

Supportive validation

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Experimental details: Figure 2 Onset and development of plaques in ARTE10 mice. A-C , Digital micrographs of parasagittal forebrain sections from homozygous transgenic mice immunohistochemically stained for beta-amyloid plaques ( dark blue-green ) at 3 (A), 8 (B), and 13 months of age (C). D-F Plaques are quantified using Acapella(tm) plaque recognition software in regions of interest on stained mouse brain sections. D , Plaque load is expressed as percentage of plaque area per total area of neocortex and hippocampus across ages in homozygous ( red ) versus hemizygous mice ( blue ). Each symbol represents one animal. The average size (E) and number of plaques per area (F) progressively increase with age in hemizygous (tg) and homozygous (tg/tg) ARTE10 mice. Data are expressed as means + SEM; * p

Supportive validation

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Experimental details: Figure 4 Human neural cultures with reduced KLC1 exhibit altered APP metabolism. (A) APP proteolytic processing by either beta-and gamma-secretases or alpha- and gamma-secretases produces sAPPbeta and Abeta (shaded dark grey) or sAPPalpha and p3 fragments, respectively. (B-C) PA6 feeder neural differentiation cultures were harvested after seven weeks and equal protein from control and shKLC1-1 cultures were analyzed using Western blots. (B) Representative immunoblots for full length APP in control and shKLC1-1 neural differentiation lysates. Results for both amino (APP-N; LN27) and carboxy terminal (APP-C) antibodies are shown. The APP carboxyl terminal cleavage fragments were not reliably detected. (C) Quantification of full length APP levels relative to NSE. (D) Levels of extracellular human Abeta peptides 38, 40 or 42 amino acids in length detected in media conditioned by control or shKLC1-1 hESC co-cultured with PA6 feeder cells for seven weeks. Human Abeta was not detected from PA6 feeder only cultures. (E) Levels of Triton X-100 soluble intracellular human Abeta-40 in control or shKLC1-1 PA6 feeder differentiation cultures aged seven weeks. Intracellular Abeta peptides 38 or 42 amino acids long were not detectable. (F) Levels of human extracellular sAPPalpha and sAPPbeta were detected in media conditioned by control or shKLC1-1 PA6 feeder cocultures aged in vitro for seven weeks. Based on n = 6 each line; *p

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Experimental details: Figure 4 Functional deficits in the HCN1 channel facilitate Abeta generation. ( A ) Quantification of endogenous murine Abeta40 and Abeta42 in the cortex of 4-month-old HCN1 +/+ , HCN1 +/- , and HCN1 -/- mice by using the sELISA system (left panels). Statistical analysis was performed using the two-tailed Mann-Whitney U -test (average +- SEM, n = 5, **p < 0.01). ( B ) Quantification of Abeta40 and Abeta42 secreted into the culture medium by N2a cells by using the sELISA system. FLAG-APP or FLAG-APP and HCN1 were transiently overexpressed in the cells with (+) or without (-) 10 muM ZD7288 (left panels). Statistical analysis was performed using one-way analysis of variance followed by Tukey's multiple comparison test (average +- SEM, n = 4, ***p < 0.001). Expression levels of APP and HCN1 was confirmed by immunoblotting (right panels in A and B ).

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Experimental details: Figure 6 HCN1 levels decrease in a age- and AD-dependent manner. ( A ) Quantification of the amount of HCN1 and actin in the brain (superior temporal gyrus) of 4-37-year-old cynomolgus monkeys by immunoblotting. Statistical analysis was performed using the two-tailed Pearson's correlation coefficient (HCN1: n = 39, r = -0.5363, ***p < 0.001; actin: n = 39, r = -0.1981, p > 0.05). ( B ) Quantification of the amount of HCN1, APP, actin, and tubulin in age-matched AD and non-AD postmortem brains (superior temporal gyrus) by immunoblotting. Statistical analysis was performed using the two-tailed Mann-Whitney U -test (non-AD: n = 13, AD: n = 12, **p < 0.01). ( C ) Comparison of postmortem time between non-AD and AD samples. Statistical analysis was performed using the two-tailed Mann-Whitney U -test (non-AD: n = 13, AD: n = 12, p = 0.8066). The correlation between postmortem time and HCN1 level was analyzed. Statistical analysis was performed using the two-tailed Pearson's correlation coefficient (n = 39, r = -0.2416, p = 0.2446). Descriptions of all subjects are provided in Table 1 .

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Experimental details: Figure 3 Features of thalamic pathology. (A) High power (100x objective) photo-micrographs of thalamic APP pathology shows large numbers of 1-2 mum diameter positive elements as well as smaller numbers of much larger spheroids (>6 mum). For comparative purposes, a number of other pathological features were examined for similarities and these were photographed at 40x. (B) Sy38 shows large spheroids of similar size to APP and significant overall loss, but no small deposits. (C) Ubiquitin shows large numbers of small 'dot-like' deposits that are similar in size but very much fewer that those observed with APP . (D) Phospho tau (AT8) labelling shows frequent apparently neuritic labelling. (E) SMI -32, neurofilament H labelling showed strong spheroid labelling in axons emerging from the medial lemniscus but much fewer spheroids in the grey matter. (F) Cathepsin D showed typical lysosomal labelling of small spherical structures in a perinuclear location, predominantly visible in neurons of the PO in NBH animals. (G) Cathepsin D in ME7 animals showed predominantly glial localization and consisted of larger structures or accumulations in perinuclear but also in some glial processes. figure 2015 British Neuropathological Society

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Experimental details: Fig 2 NaCl treatment did not affect gamma secretase. (A) FACS analysis showed the Annexin V-FITC/ PI staining of HEK293 cells treated with normal (up) or NaCl (low) medium. Western blots (B) and their quantification (C) showed the protein levels of APP and gamma secretase components (PS1, NCT, PEN2) in the lysate of HEK293 cells overexpressing APP after normal or NaCl treatment (n = 3). (D) Enzyme activity assay showed the gamma secretase activity of HEK293 cell lysate after treatment with normal, NaCl medium or gamma secretase inhibitor-L685,458 (n = 3). Data were presented as means +- s.e.m. of indicated numbers of independent experiments.

Supportive validation

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Experimental details: Fig. 4 hNSC transplantation decreases brain intracellular soluble Abeta42 levels by regulating BACE1 in NSE/APPsw transgenic mice. a Photomicrographs of Abeta plaque (6E10) staining in hNSC-injected (NSC) and vehicle-injected (Veh) transgenic mice. Scale bar, 1 mm. b The number of Abeta plaques per unit area (left) and the percent area of the Abeta plaque load (right) between hNSC graft (NSC) and vehicle injection (Veh; n = 3 per group). c - l Representative images of Abeta42 immunostaining in the brains of vehicle-injected wild-type mice (WT-Veh; c ), and of vehicle-injected (APP-Veh; d ) and hNSC-injected (APP-NSC; e ) transgenic mice. Abeta42 immunostaining of each group in the pyramidal cell layer of the hippocampal CA1 region (HIPP; f - h ) and the posterior parietal cortex (CTX; i - k ). The relative levels of Abeta42 immunostaining in vehicle-injected (Veh) and hNSC-injected (NSC) transgenic mice ( n = 4 per group; l ). Scale bar, 100 mum ( e and h ) and 50 mum ( k ). m Levels of detergent-soluble (Sol) Abeta40/42 and detergent-insoluble (Insol) Abeta40/42 in the brains of hNSC-injected (NSC) and vehicle-injected (Veh) transgenic mice using ELISA kits ( n = 4 per group). n and o Relative levels of BACE1 (NSC, n = 6; Veh, n = 5; n ) and APP CTF-beta/CTF-alpha (NSC, n = 4; Veh, n = 3; o ) using western blot analyses in brains of hNSC-injected (NSC) and vehicle-injected (Veh) transgenic mice. p APPsw-expressing SK-N-MC cells treated with hNSC-derived (NSC) and fibroblast-

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Experimental details: Figure 3 Augmentation of membrane cholesterol levels increased PLC beta 1 and PLC beta 3 expressions. (a) Representative western blotting results showed specific increase of PLC beta 1 and PLC beta 3 expressions from APP-transfected HeLa cells. Cells were incubated with 75 mu M water-soluble cholesterol for the indicated times. Membrane and cytosol fractions were obtained as described in Materials and Methods. Similar results were obtained from 5 different experiments. Note that PLC beta 1 and PLC beta 3 expressions were increased in time-dependent manner by cholesterol from membrane fraction but not from cytosol fraction. In contrast, PLC gamma 2 expression was not changed by cholesterol. beta -tubulin was used to confirm the amount of proteins loaded. (b, c) Bars correspond to the densitometric analysis of PLC beta 1 and PLC beta 3 expressions from membrane and cytosol fractions ( n = 5). * P < 0.05; ** P < 0.01.

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Experimental details: Figure 3 TRPC6 interacted with APP (C99). ( a ) Immunocytochemical analysis of HEK293 cells transfected with APP-Myc and TRPC6-HA for 3 days. EEA1, Calnexin and GM130 are markers for early endosome, ER and Golgi, respectively. Arrow heads indicated the co-localized signals. Scale bar, 20 mum. ( b ) Immunoblots of indicated proteins after fractionation of mouse brain lysates. Bip and Cathepsin D are markers for ER and lysosome, respectively. Arrow indicated alignment of two gels with 11 lanes running in parallel. ( c ) Wild-type mouse brain lysates precipitated with the antibody against APP, PS1 or Notch, and immunoblotted with indicated antibodies. ( d ) Lysates of HEK293APP stable cells transfected with TRPC5 or 6 for 2 days precipitated with APP antibody, and immunoblotted with indicated antibodies. ( e ) Lysates of HEK293TRPC6 stable cells transfected with C99-Myc or NotchDeltaE-Myc for 1 day, treated with 10 muM L685,458 for 12 h and then precipitated with Myc or TRPC6 antibody, and immunoblotted with indicated antibodies. ( f ) Lysates of HEK293C99-Myc stable cells transfected with PS1-HA together with YFP or TRPC6 for 2 days and precipitated with Myc or HA antibody, and immunoblotted with indicated antibodies. Lower, quantification of the reciprocal precipitated protein levels of C99 and PS1 ( n =4). CTRL, transfection with YFP. Data were presented as means+-s.e.m. of indicated numbers of independent experiments. Two-tailed Student's t -test was performed. * P

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Experimental details: Figure 4 TAT-TM2 reduced Abeta levels in vitro and in vivo . ( a ) ELISA examination of Abeta levels in the medium of HEK293APP cells transfected with TRPC6 or its mutants within second transmembrane domain (TM2) for 2 days ( n =3-6). ( b ) Lysates of HEK293APP stable cells transfected with C6-mut1 for 2 days precipitated with APP antibody and immunoblotted with indicated antibodies. ( c ) The Abeta levels in the medium of HEK293APP cells treated with 2 muM indicated peptides for 12 h ( n =3-6). ( d ) Lysates of HEK293APP stable cells incubated with 5 muM TAT-biotin or TAT-TM2-bitoin for 6 h precipitated with avidin beads and immunoblotted with indicated antibodies. ( e ) ELISA examination in a blinded manner of Abeta levels in the brain lysates of 5 month male APP/PS1 mice intra-peritoneally injected with 200 mul 2.5 mM TAT or TAT-TM2 for 3 h ( n =8 mice). ( f ) The Abeta levels generated in the in vitro purified C99 cleavage assay treated with 2 muM of indicated peptides for 2 h ( n =4). ( g ) Abeta levels in the medium of COS7C99 stable cells treated with 5 muM TAT-TM2 for 12 h ( n =3-4). ( h ) Immunoblots of AICD in HEK293C99 cells treated with 5 muM TAT-TM2 for 12 h or NICD in HEK293APP cells transfected with NotchE-myc for 1 day and then treated with 5 muM TAT-TM2 for 12 h. ( i ) Quantification of the ratio of AICD/C99 or NICD/NotchDeltaE ( n =3-4). CTRL, transfection with YFP . Data were presented as means+-s.e.m. of indicated numbers of independent experiments. Two-ta

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Experimental details: Figure 5 FcRn expression in trisomic fibroblasts and impact of APP on FCGRT expression. ( A ) FCGRT mRNA relative fold expression (left), and FcRn protein expression (right) in trisomic (DS-1, -2, and -3) and diploid fibroblasts (NDS-1, -2, and -3) from sex and age-matched donors. Each bar represents the mean +- SD from two separate measurements performed in quadruplicate. Upper right panel: densitometric analysis of FcRn bands normalized to tubulin. ( B ) Linear regression analysis of FCGRT versus APP mRNA relative expression in diploid and trisomic fibroblasts (n = 8 cell lines). Each point represents the mean from two separate measurements performed in quadruplicate. ( C ) FCGRT mRNA relative expression in diploid (left panel, line AG07095, NDS-2) and trisomic fibroblasts (right panel, line AG06922, DS-2) analyzed 96 h post-transfection with a non-sense siRNA (NS-siRNA, control) or a siRNA pool against APP ( APP siRNA). ( D , E ) FCGRT mRNA relative expression (left panel), APP-GFP expression detected by fluorescence microscopy (upper middle panel), and FcRn and APP protein expression detected by immunoblotting (right panel) in AG07095 (NDS-2. D ) and HepG2 ( E ). Lower middle panel: densitometric analysis of FcRn bands normalized to tubulin. Cells were analyzed 48 h post-transfection with an empty vector (EV, control) or an APP-GFP plasmid. Each bar represents the mean +- SD from at least two measurements performed in quadruplicate. ***P < 0.001, **P < 0.01, *P < 0.05, St

Supportive validation

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Experimental details: Figure 6 Neuronal, APP and synaptic pathology in the principal trigeminal nucleus. (A-F) Micrographs illustrating NeuN labelling in PrTNdm and PrTNvl of NBH (A,D) and ME7 (B,E) animals, respectively. Masks used to quantify the number of neurons per area, generated in ImageJ software, are shown in supplemental material. ME7 induced significant neuronal loss in PrTNvl (F) but not in PrTNdm (C). * t -test, P < 0.01. n = 7 ME7 , n = 6 NBH . Scale bar: 100 mum. (G-I) Immunolabelling for APP in the PrTN of NBH (G) and ME7 (H). Greyscale images were treated to differentiate brown and blue labelling (see methods) and then thresholded in ImageJ software in order to quantifty APP deposits only. The percentage of APP -labelled area in the PrTN of NBH and ME7 animals ( n = 4 in each group) is shown in (I). * t -test, P < 0.05. (J-L) Immunolabelling for Sy38 in PrTN of NBH (J) and ME7 animals ( K ). The regions of PrTN : ventrolateral part (in white) and dorsomedial part (in black) are delineated in (J). Contrast indices are shown for synaptophysin transmittance measurements in PrTNvl and PrTNdm of NBH (J) and ME7 ( K ) groups. Statistically significant reduction of synaptic density was found in PrTNvl , but not in PrTNdm (L). * t -test, P < 0.01. PrTNVL , principal trigeminal nucleus, ventrolateral part; PrTNdm, principal trigeminal nucleus, dorsomedial part. n = 10 ME7 and 6 NBH . Scale bar: 1 mm. All data are shown as mean +- SEM . figure 2015 British Neuropathological Society

Supportive validation

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Experimental details: Fig. 1 APP is highly expressed in macrophages and microglia and binds HIV-1 Gag. a APP processing through amyloidogenic, non-amyloidogenic and eta-secretase pathways involves alpha-, beta-, gamma- and eta-secretases. The Abeta peptide resulting in toxic amyloid oligomers and plaques is generated by sequential cleavages by beta- and gamma-secretases via the amyloidogenic pathway (central). b Human APP 770 (APP-Flag) binds HIV-1 Gag (Gag-HA) in anti-APP co-IP from transfected 293T cells. c Endogenous APP and Gag colocalize in CHME3 cells infected with HIV-1 carrying vesicular stomatitis virus G (VSV-G) envelope glycoprotein at 16 h post infection (h.p.i). Nuclei were stained with Hoechst (blue). All images were obtained using a 100x oil objective of a spinning disk confocal microscope. Scale bar = 10 mum. d Quantification of APP and Gag as determined by Pearson's coefficient in at least 10 random fields of view from samples as in c , shown as Mean +- SEM. The Pearson's correlation coefficients were r = 0.512 +- 0.02. e Endogenous APP levels in glioblastoma (U87), normal human dermal fibroblasts (NHDF), microglia (CHME3), 293T and differentiated THP-1 cells. Molecular weight markers (in kDa) are shown to the right of WBs

Supportive validation

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Experimental details: Fig. 4 Treating SIV-infected macaques with maraviroc (MVC) significantly reduced CD68 immunostaining (a) in the basal ganglia compared with untreated SIV-infected macaques (open circles) to levels equivalent to uninfected macaques (closed circles). SIV-infected macaques treated with MVC had significantly reduced APP immunostaining in the corpus callosum compared with untreated SIV-infected macaques (circles) to levels unchanged from untreated SIV-infected macaques. Bars represent median values; Mann-Whitney test.