aβ42  (Thermo Fisher)


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    Name:
    Amyloid beta 42 Human ELISA Kit
    Description:
    The Human Beta Amyloid 1 42 Aβ42 ELISA research use only kit is to be used for the quantitative determination of human Aβ42 in samples e g cell culture supernatants tissue homogenates cerebrospinal fluid CSF etc using 96 well plates and a microplate reader The assay recognizes both natural and synthetic forms of human Aβ42 The anti human Aβ42 antibody used in this kit is capable of selectively detecting Aβ42 and not Aβ40 Aβ43 Performance characteristics • Sensitivity 10 pg mL • Standard curve range 15 6 1 000 pg mL • Sample types cell culture supernatants tissue homogenates cerebrospinal fluid CSF • Species cross reactivity human • Sample volume 50 μL prediluted • Total assay incubation time 4 hrs Principle of the method The Human Aβ42 kit is a solid phase sandwich enzyme linked immunosorbent assay ELISA A monoclonal antibody specific for the NH2 terminus of human Aβ has been coated onto the wells of the microtiter strips provided During the first incubation standards of known human Aβ42 content controls and unknown samples are pipetted into the wells and co incubated with a rabbit antibody specific for the COOH terminus of the 1 42 Aβ sequence This COOH terminal sequence is created upon cleavage of the analyzed precursor After washing bound rabbit antibody is detected by the addition of a horseradish peroxidase labeled anti rabbit antibody After a second incubation and washing to remove all of the unbound enzyme a substrate solution is added which is acted upon by the bound enzyme to produce color The intensity of this colored product is directly proportional to the concentration of human Aβ42 present in the original specimen Background information Beta amyloid peptide Aβ is a 40 to 43 amino acid peptide cleaved from amyloid precursor protein by β secretase e g BACE and a putative γ gamma secretase Increased release of the longer forms of Aβ peptide Aβ42 or Aβ43 which have a greater tendency to aggregate than Aβ40 occurs in individuals expressing certain genetic mutations expressing certain ApoE alleles or may involve other still undiscovered factors Many researchers theorize that it is this increased release of Aβ42 Aβ43 which leads to the abnormal deposition of Aβ Each Human Aβ42 ELISA kit is validated for sensitivity specificity precision and lot to lot consistency See product insert for more information on validation Related links Learn more about ELISA kits Learn more about other immunoassays Please refer to the ELISA protocol for specific reference citations
    Catalog Number:
    KHB3441
    Price:
    None
    Category:
    Kits and Assays
    Applications:
    Protein Assays and Analysis|Protein Biology
    Buy from Supplier


    Structured Review

    Thermo Fisher aβ42
    Complement activation mediates microglial phagocytosis of Aβ in vitro . A , Representative images of fluorescent FAM-labeled <t>Aβ42</t> peptide uptake in primary wild-type microglial cultures treated with vehicle (PBS) or 10 μg/ml C3 for
    The Human Beta Amyloid 1 42 Aβ42 ELISA research use only kit is to be used for the quantitative determination of human Aβ42 in samples e g cell culture supernatants tissue homogenates cerebrospinal fluid CSF etc using 96 well plates and a microplate reader The assay recognizes both natural and synthetic forms of human Aβ42 The anti human Aβ42 antibody used in this kit is capable of selectively detecting Aβ42 and not Aβ40 Aβ43 Performance characteristics • Sensitivity 10 pg mL • Standard curve range 15 6 1 000 pg mL • Sample types cell culture supernatants tissue homogenates cerebrospinal fluid CSF • Species cross reactivity human • Sample volume 50 μL prediluted • Total assay incubation time 4 hrs Principle of the method The Human Aβ42 kit is a solid phase sandwich enzyme linked immunosorbent assay ELISA A monoclonal antibody specific for the NH2 terminus of human Aβ has been coated onto the wells of the microtiter strips provided During the first incubation standards of known human Aβ42 content controls and unknown samples are pipetted into the wells and co incubated with a rabbit antibody specific for the COOH terminus of the 1 42 Aβ sequence This COOH terminal sequence is created upon cleavage of the analyzed precursor After washing bound rabbit antibody is detected by the addition of a horseradish peroxidase labeled anti rabbit antibody After a second incubation and washing to remove all of the unbound enzyme a substrate solution is added which is acted upon by the bound enzyme to produce color The intensity of this colored product is directly proportional to the concentration of human Aβ42 present in the original specimen Background information Beta amyloid peptide Aβ is a 40 to 43 amino acid peptide cleaved from amyloid precursor protein by β secretase e g BACE and a putative γ gamma secretase Increased release of the longer forms of Aβ peptide Aβ42 or Aβ43 which have a greater tendency to aggregate than Aβ40 occurs in individuals expressing certain genetic mutations expressing certain ApoE alleles or may involve other still undiscovered factors Many researchers theorize that it is this increased release of Aβ42 Aβ43 which leads to the abnormal deposition of Aβ Each Human Aβ42 ELISA kit is validated for sensitivity specificity precision and lot to lot consistency See product insert for more information on validation Related links Learn more about ELISA kits Learn more about other immunoassays Please refer to the ELISA protocol for specific reference citations
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    Images

    1) Product Images from "Astrocyte-Microglia Cross Talk through Complement Activation Modulates Amyloid Pathology in Mouse Models of Alzheimer's Disease"

    Article Title: Astrocyte-Microglia Cross Talk through Complement Activation Modulates Amyloid Pathology in Mouse Models of Alzheimer's Disease

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2117-15.2016

    Complement activation mediates microglial phagocytosis of Aβ in vitro . A , Representative images of fluorescent FAM-labeled Aβ42 peptide uptake in primary wild-type microglial cultures treated with vehicle (PBS) or 10 μg/ml C3 for
    Figure Legend Snippet: Complement activation mediates microglial phagocytosis of Aβ in vitro . A , Representative images of fluorescent FAM-labeled Aβ42 peptide uptake in primary wild-type microglial cultures treated with vehicle (PBS) or 10 μg/ml C3 for

    Techniques Used: Activation Assay, In Vitro, Labeling

    2) Product Images from "The blockage of the Nogo/NgR signal pathway in microglia alleviates the formation of Aβ plaques and tau phosphorylation in APP/PS1 transgenic mice"

    Article Title: The blockage of the Nogo/NgR signal pathway in microglia alleviates the formation of Aβ plaques and tau phosphorylation in APP/PS1 transgenic mice

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0522-x

    CM from BV-2 microglia activated by Nogo/NgR pathway increased the production and aggregation of Aβ. After exposing for CM of BV-2 microglia for 24 h, ELISA was performed to detect the production of Aβ40 ( a ) and Aβ42 ( b ) and the secretion of Aβ40 ( c ) and Aβ42 ( d ). Values were reported as mean ± SD. The aggregation of Aβ ( e ) and the depolymerization of fibrillar Aβ ( f ) were determined using ThT binding assay. Values were reported as mean ± SD, as a percentage of values determined in PBS group (control, 100 %). *p
    Figure Legend Snippet: CM from BV-2 microglia activated by Nogo/NgR pathway increased the production and aggregation of Aβ. After exposing for CM of BV-2 microglia for 24 h, ELISA was performed to detect the production of Aβ40 ( a ) and Aβ42 ( b ) and the secretion of Aβ40 ( c ) and Aβ42 ( d ). Values were reported as mean ± SD. The aggregation of Aβ ( e ) and the depolymerization of fibrillar Aβ ( f ) were determined using ThT binding assay. Values were reported as mean ± SD, as a percentage of values determined in PBS group (control, 100 %). *p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

    Inhibition of the Nogo/NgR pathway reduced Aβ plaque deposition in APP/PS1 mice. a, b Mice brain sections from APP/PS1 mice were stained with thioflavin S and calculated as Aβ-positive area fraction in the hippocampal and cortical regions at 8 months of age. Bar = 100 μM. Values were reported as mean ± SD, as a percentage of values determined in the vehicle group (control, 100 %). The Aβ plaque in the brain was estimated after immunohistochemistry with Aβ antibody (6E10), and the position area fraction of the hippocampal and cortical regions of brain tissues were calculated ( c, d ). Bar = 100 μM. Values were reported as mean ± SD, as a percentage of values determined in the vehicle group (control, 100 %). e–h The cortex or hippocampus of APP/PS1 mice was homogenized and separated into TBS-, TBST-, and guanidine-soluble fractions. The proportions of Aβ42 ( e, f ) and Aβ40 ( g, h ) were measured using ELISA. e , g The cortex of APP/PS1 mice; f , h The hippocampus of APP/PS1 mice. Values were reported as mean ± SD. *p
    Figure Legend Snippet: Inhibition of the Nogo/NgR pathway reduced Aβ plaque deposition in APP/PS1 mice. a, b Mice brain sections from APP/PS1 mice were stained with thioflavin S and calculated as Aβ-positive area fraction in the hippocampal and cortical regions at 8 months of age. Bar = 100 μM. Values were reported as mean ± SD, as a percentage of values determined in the vehicle group (control, 100 %). The Aβ plaque in the brain was estimated after immunohistochemistry with Aβ antibody (6E10), and the position area fraction of the hippocampal and cortical regions of brain tissues were calculated ( c, d ). Bar = 100 μM. Values were reported as mean ± SD, as a percentage of values determined in the vehicle group (control, 100 %). e–h The cortex or hippocampus of APP/PS1 mice was homogenized and separated into TBS-, TBST-, and guanidine-soluble fractions. The proportions of Aβ42 ( e, f ) and Aβ40 ( g, h ) were measured using ELISA. e , g The cortex of APP/PS1 mice; f , h The hippocampus of APP/PS1 mice. Values were reported as mean ± SD. *p

    Techniques Used: Inhibition, Mouse Assay, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

    3) Product Images from "A Becn1 mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in Alzheimer's disease"

    Article Title: A Becn1 mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in Alzheimer's disease

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006962

    An autophagy-inducing compound ML246 reduces amyloid load in an autophagy-dependent manner in vitro and in vivo. (A) Chemical structure of ML246. (B) Dot-blot assays (left) and quantification (right) of secreted Aβ42 levels in conditioned media of HEK293 cells stably expressing APP treated with vehicle (DMSO) or ML246 for 24 h, immunostained with anti-Aβ42 antibody. Cells were transfected with non-targeting control (NC) or ATG7 siRNA 24 h prior to ML246 treatment. Results are quantified from 4 independent experiments. (C) Representative images (left) and quantification (right) of TUNEL signals (red) in WT primary cortical neurons treated with conditioned media from ( B ) for 24 h. Nuclei were stained with DAPI. Scale bar, 100 μm. N = 10 fields (each field containing 20–30 neurons). (D, E) Representative images (upper) and quantification (lower) of dot-blot assays on soluble (D) and insoluble (E) Aβ42 levels in brain samples of 6-month old 5XFAD and 5XFAD Becn1 +/- KO mice after 5 weeks of ML246 treatment, immunostained with anti-Aβ42 antibody. Total protein loading was labeled by Ponceau S. Triplicate experiments from 4–5 mice in each group were shown. Results represent mean ± s.e.m. NS, not significant; *, P
    Figure Legend Snippet: An autophagy-inducing compound ML246 reduces amyloid load in an autophagy-dependent manner in vitro and in vivo. (A) Chemical structure of ML246. (B) Dot-blot assays (left) and quantification (right) of secreted Aβ42 levels in conditioned media of HEK293 cells stably expressing APP treated with vehicle (DMSO) or ML246 for 24 h, immunostained with anti-Aβ42 antibody. Cells were transfected with non-targeting control (NC) or ATG7 siRNA 24 h prior to ML246 treatment. Results are quantified from 4 independent experiments. (C) Representative images (left) and quantification (right) of TUNEL signals (red) in WT primary cortical neurons treated with conditioned media from ( B ) for 24 h. Nuclei were stained with DAPI. Scale bar, 100 μm. N = 10 fields (each field containing 20–30 neurons). (D, E) Representative images (upper) and quantification (lower) of dot-blot assays on soluble (D) and insoluble (E) Aβ42 levels in brain samples of 6-month old 5XFAD and 5XFAD Becn1 +/- KO mice after 5 weeks of ML246 treatment, immunostained with anti-Aβ42 antibody. Total protein loading was labeled by Ponceau S. Triplicate experiments from 4–5 mice in each group were shown. Results represent mean ± s.e.m. NS, not significant; *, P

    Techniques Used: In Vitro, In Vivo, Dot Blot, Stable Transfection, Expressing, Transfection, TUNEL Assay, Staining, Mouse Assay, Labeling

    Autophagosomal sequestration of Aβ42 in brain of autophagy-hyperactive mice. (Left) Scheme of immunoisolation of autophagosomes from brain of 12-week old 5XFAD Becn1 FA/FA mice expressing GFP-LC3. Briefly, post-nucleus extracts of the brain lysates was obtained by centrifugation at a low speed of 1,000 xg. Autophagosomes were enriched by centrifugation at a high speed of 20,000 xg, and pulled down by an anti-GFP antibody using magnetic beads. (Right) Western blot detection of Aβ42 fibrillar and oligomeric species inside autophagosomes immunoprecipitated by GFP antibody as in the scheme. A known autophagy cargo p62 serves as a positive control, and a cytosolic enzyme GAPDH is a negative control.
    Figure Legend Snippet: Autophagosomal sequestration of Aβ42 in brain of autophagy-hyperactive mice. (Left) Scheme of immunoisolation of autophagosomes from brain of 12-week old 5XFAD Becn1 FA/FA mice expressing GFP-LC3. Briefly, post-nucleus extracts of the brain lysates was obtained by centrifugation at a low speed of 1,000 xg. Autophagosomes were enriched by centrifugation at a high speed of 20,000 xg, and pulled down by an anti-GFP antibody using magnetic beads. (Right) Western blot detection of Aβ42 fibrillar and oligomeric species inside autophagosomes immunoprecipitated by GFP antibody as in the scheme. A known autophagy cargo p62 serves as a positive control, and a cytosolic enzyme GAPDH is a negative control.

    Techniques Used: Mouse Assay, Expressing, Centrifugation, Magnetic Beads, Western Blot, Immunoprecipitation, Positive Control, Negative Control

    ML246 and voluntary exercise decrease cerebral amyloid plaques and ameliorate memory deficits in 5XFAD AD mice. (A, B) Representative images (upper) and quantification (lower) of dot-blot assays on soluble (A) and insoluble (B) Aβ42 levels in brain samples of 6-month old 5XFAD and 5XFAD Becn1 +/- KO mice after 4 months of voluntary running, immunostained with anti-Aβ42 antibody. Total protein loading was labeled by Ponceau S. Triplicate experiments from 4–5 mice in each group were shown. (C) Representative images (left) and quantification (right) of amyloid deposits stained by Thioflavin S in brain of 6-month old 5XFAD mice, and 5XFAD mice subject to 5 weeks of ML246 treatment or 4 months of voluntary exercise. Scale bar: 500 μm. Results represent mean ± s.e.m. N = 6–8. *, P
    Figure Legend Snippet: ML246 and voluntary exercise decrease cerebral amyloid plaques and ameliorate memory deficits in 5XFAD AD mice. (A, B) Representative images (upper) and quantification (lower) of dot-blot assays on soluble (A) and insoluble (B) Aβ42 levels in brain samples of 6-month old 5XFAD and 5XFAD Becn1 +/- KO mice after 4 months of voluntary running, immunostained with anti-Aβ42 antibody. Total protein loading was labeled by Ponceau S. Triplicate experiments from 4–5 mice in each group were shown. (C) Representative images (left) and quantification (right) of amyloid deposits stained by Thioflavin S in brain of 6-month old 5XFAD mice, and 5XFAD mice subject to 5 weeks of ML246 treatment or 4 months of voluntary exercise. Scale bar: 500 μm. Results represent mean ± s.e.m. N = 6–8. *, P

    Techniques Used: Mouse Assay, Dot Blot, Labeling, Staining

    Becn1 FA/FA mutation ameliorates cerebral Aβ accumulation, memory deficits and mortality of Alzheimer’s mouse models. (A-B) Dot-blot assays and quantification of soluble (A) and insoluble (B) Aβ42 levels in homogenated brain samples of 6-month old 5XFAD Becn1 +/+ and 5XFAD Becn1 FA/FA mice, immunostained with anti-Aβ42 antibody. Total protein loading was labeled by Ponceau S. Triplicate experiments from 6 mice in each group were shown. ***, P
    Figure Legend Snippet: Becn1 FA/FA mutation ameliorates cerebral Aβ accumulation, memory deficits and mortality of Alzheimer’s mouse models. (A-B) Dot-blot assays and quantification of soluble (A) and insoluble (B) Aβ42 levels in homogenated brain samples of 6-month old 5XFAD Becn1 +/+ and 5XFAD Becn1 FA/FA mice, immunostained with anti-Aβ42 antibody. Total protein loading was labeled by Ponceau S. Triplicate experiments from 6 mice in each group were shown. ***, P

    Techniques Used: Mutagenesis, Dot Blot, Mouse Assay, Labeling

    4) Product Images from "Biomarkers of Alzheimer Disease in Children with Obstructive Sleep Apnea: Effect of Adenotonsillectomy"

    Article Title: Biomarkers of Alzheimer Disease in Children with Obstructive Sleep Apnea: Effect of Adenotonsillectomy

    Journal: Sleep

    doi: 10.5665/sleep.5838

    Individual and boxplot graphs of apnea-hypopnea index (AHI) (A) , oxyhemoglobin desaturation 3% index (ODI 3%) (B) , respiratory arousal index (RAI) (C) , serum levels of Aβ42 (D) and PS1 (E) in 24 children with OSA before and after adenotonsillectomy
    Figure Legend Snippet: Individual and boxplot graphs of apnea-hypopnea index (AHI) (A) , oxyhemoglobin desaturation 3% index (ODI 3%) (B) , respiratory arousal index (RAI) (C) , serum levels of Aβ42 (D) and PS1 (E) in 24 children with OSA before and after adenotonsillectomy

    Techniques Used:

    Scattergrams of Aβ42 (A) and PS1 (B) individual serum levels plotted against apnea-hypopnea index (AHI), oxyhemoglobin desaturation 3% index (ODI 3%), and respiratory arousal index (RAI) in 286 children. Linear regression lines are shown as solid
    Figure Legend Snippet: Scattergrams of Aβ42 (A) and PS1 (B) individual serum levels plotted against apnea-hypopnea index (AHI), oxyhemoglobin desaturation 3% index (ODI 3%), and respiratory arousal index (RAI) in 286 children. Linear regression lines are shown as solid

    Techniques Used:

    Individual and boxplot graphs of plasma levels of Aβ42 (A) and PS1 (B) in obese children (OB), children with OSA, children who are obese and suffer from OSA (OSA+OB) and controls (CO). Statistically significant increases in Aβ42 and PS1
    Figure Legend Snippet: Individual and boxplot graphs of plasma levels of Aβ42 (A) and PS1 (B) in obese children (OB), children with OSA, children who are obese and suffer from OSA (OSA+OB) and controls (CO). Statistically significant increases in Aβ42 and PS1

    Techniques Used:

    5) Product Images from "The biochemical aftermath of anti-amyloid immunotherapy"

    Article Title: The biochemical aftermath of anti-amyloid immunotherapy

    Journal: Molecular Neurodegeneration

    doi: 10.1186/1750-1326-5-39

    Gray matter of UCSD immunized cases separated by HPLC (C8 reverse-phase) . The figure shows the HPLC chromatogram fractions that were investigated by Western blotting developed with anti-Aβ40 and anti-Aβ42 and CT9APP antibodies. The diagonal-hyphenated line represents the acetonitrile gradient. Lanes 10 contain the standards Aβ40 or Aβ42 synthetic peptides. A) and B), correspond to the neuropathologically confirmed AD cases # 19 and # 20, respectively. C) corresponds to the neuropathologically confirmed hippocampal sclerosis. m, Aβ monomer; d, Aβ dimer; t, Aβ trimer.
    Figure Legend Snippet: Gray matter of UCSD immunized cases separated by HPLC (C8 reverse-phase) . The figure shows the HPLC chromatogram fractions that were investigated by Western blotting developed with anti-Aβ40 and anti-Aβ42 and CT9APP antibodies. The diagonal-hyphenated line represents the acetonitrile gradient. Lanes 10 contain the standards Aβ40 or Aβ42 synthetic peptides. A) and B), correspond to the neuropathologically confirmed AD cases # 19 and # 20, respectively. C) corresponds to the neuropathologically confirmed hippocampal sclerosis. m, Aβ monomer; d, Aβ dimer; t, Aβ trimer.

    Techniques Used: High Performance Liquid Chromatography, Western Blot

    6) Product Images from "Hydrogen Sulfide Inhibits A2A Adenosine Receptor Agonist Induced ?-Amyloid Production in SH-SY5Y Neuroblastoma Cells via a cAMP Dependent Pathway"

    Article Title: Hydrogen Sulfide Inhibits A2A Adenosine Receptor Agonist Induced ?-Amyloid Production in SH-SY5Y Neuroblastoma Cells via a cAMP Dependent Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088508

    Effect of NaHS on mRNA expression of AC isoforms and AC activity. A–B : Representative gels (A) and histogram (B) demonstrating the effect of pretreatment with NaHS (100 µM, 12 hours) attenuated the effects of HENECA (100 nM, 24 hours) on mRNA expressions of AC isoforms. C : Effect of NaHS (100 µM) on AC activity stimulated by forskolin (20 µM). D : Effect of NaHS (100 µM) on Aβ42 production in SH-SY5Y cells preincubated with AC antagonist, SQ 22536 (300 µM). Control values were adjusted to 100%. Data are given as means ± S.E.M, n = 4–6. ## p
    Figure Legend Snippet: Effect of NaHS on mRNA expression of AC isoforms and AC activity. A–B : Representative gels (A) and histogram (B) demonstrating the effect of pretreatment with NaHS (100 µM, 12 hours) attenuated the effects of HENECA (100 nM, 24 hours) on mRNA expressions of AC isoforms. C : Effect of NaHS (100 µM) on AC activity stimulated by forskolin (20 µM). D : Effect of NaHS (100 µM) on Aβ42 production in SH-SY5Y cells preincubated with AC antagonist, SQ 22536 (300 µM). Control values were adjusted to 100%. Data are given as means ± S.E.M, n = 4–6. ## p

    Techniques Used: Expressing, Activity Assay

    Effect of NaHS on Aβ42 production involved cAMP signaling pathway. A : Dose-dependent effect of HENECA on cAMP production in SH-SY5Y cells expressing APPswe. B : Concentration-dependent effect of NaHS (10–200 µM, 12 hours) on HENECA (100 nM, 24 hours)-stimulated cAMP upregulation. C–D : Effects of NaHS (100 µM) on cAMP (C) and Aβ42 production (D) in cells treated with forskolin (20 µM) and/or IBMX (100 µM). The intracellular cAMP and Aβ42 levels in conditioned media were measured by sandwich ELISA kits. Control values were adjusted to 100% for Aβ42 levels measurement. Data are given as means ± S.E.M, n = 6. ### p
    Figure Legend Snippet: Effect of NaHS on Aβ42 production involved cAMP signaling pathway. A : Dose-dependent effect of HENECA on cAMP production in SH-SY5Y cells expressing APPswe. B : Concentration-dependent effect of NaHS (10–200 µM, 12 hours) on HENECA (100 nM, 24 hours)-stimulated cAMP upregulation. C–D : Effects of NaHS (100 µM) on cAMP (C) and Aβ42 production (D) in cells treated with forskolin (20 µM) and/or IBMX (100 µM). The intracellular cAMP and Aβ42 levels in conditioned media were measured by sandwich ELISA kits. Control values were adjusted to 100% for Aβ42 levels measurement. Data are given as means ± S.E.M, n = 6. ### p

    Techniques Used: Expressing, Concentration Assay, Sandwich ELISA

    Effect of NaHS on activities of β- and γ-secretases. A–B : Representative gel (A) and quantitative analysis (B) showing NaHS (25–100 µM, 12 hours) and HENECA (100 nM, 24 hours) failed to affect β-CTF (C99) expression in SH-SY5Y cells. The membrane fractions were analysed by western blot with antibody against c-terminal fragment of APP (C99) or β-actin. C–D :Effect of NaHS (100 µM) and a γ-secretase inhibitor, DAPT (1 µM, 1 hour) on HENECA (100 nM)-stimulated γ-secretase activity (C) and Aβ42 formation (D) in SH-SY5Y cells expressing APPswe. E : Effect of NaHS (100 µM) on γ-secretase activity in SH-SY5Y cells preincubated with AC antagonist, SQ 22536 (300 µM). Data are given as means ± S.E.M, n = 4–6. ### p
    Figure Legend Snippet: Effect of NaHS on activities of β- and γ-secretases. A–B : Representative gel (A) and quantitative analysis (B) showing NaHS (25–100 µM, 12 hours) and HENECA (100 nM, 24 hours) failed to affect β-CTF (C99) expression in SH-SY5Y cells. The membrane fractions were analysed by western blot with antibody against c-terminal fragment of APP (C99) or β-actin. C–D :Effect of NaHS (100 µM) and a γ-secretase inhibitor, DAPT (1 µM, 1 hour) on HENECA (100 nM)-stimulated γ-secretase activity (C) and Aβ42 formation (D) in SH-SY5Y cells expressing APPswe. E : Effect of NaHS (100 µM) on γ-secretase activity in SH-SY5Y cells preincubated with AC antagonist, SQ 22536 (300 µM). Data are given as means ± S.E.M, n = 4–6. ### p

    Techniques Used: Expressing, Western Blot, Activity Assay

    Effect of NaHS on Aβ42 production involved PKA and CREB. A : Effect of HENECA (100 nM) on Aβ42 formation was abolished by a PKA inhibitor, H89 (5, 10 and 15 µM). B–C : Representative gel (B) and histogram (C) depicting that pretreatment with NaHS (100 µM, 12 hours) attenuated the effects of HENECA (100 nM, 24 hours) on phosphorylation of CREB. Control values were adjusted to 100%. Data are given as means ± S.E.M, n = 4–6. ### p
    Figure Legend Snippet: Effect of NaHS on Aβ42 production involved PKA and CREB. A : Effect of HENECA (100 nM) on Aβ42 formation was abolished by a PKA inhibitor, H89 (5, 10 and 15 µM). B–C : Representative gel (B) and histogram (C) depicting that pretreatment with NaHS (100 µM, 12 hours) attenuated the effects of HENECA (100 nM, 24 hours) on phosphorylation of CREB. Control values were adjusted to 100%. Data are given as means ± S.E.M, n = 4–6. ### p

    Techniques Used:

    Effect of NaHS on Aβ42 production and cell viability in SH-SY5Y cells expressing APPswe. A : Concentration-dependent effect of HENECA (10–200 nM, 24 hours) on Aβ42 production. B–C : Dose-dependent effect of NaHS (10–200 µM, 12 hours) on Aβ42 formation in the presence (B) and absence (C) of HENECA (100 nM, 24 hours). D–E : MTT assay showing the effect of NaHS alone at 10–200 µM (D) or HENECA alone at 10–200 nM (E) on cell viability of SH-SY5Y cells. Aβ42 levels in conditioned media were measured by sandwich ELISA kit. Control values were adjusted to 100%. Data are given as means ± S.E.M, n = 6. ## p
    Figure Legend Snippet: Effect of NaHS on Aβ42 production and cell viability in SH-SY5Y cells expressing APPswe. A : Concentration-dependent effect of HENECA (10–200 nM, 24 hours) on Aβ42 production. B–C : Dose-dependent effect of NaHS (10–200 µM, 12 hours) on Aβ42 formation in the presence (B) and absence (C) of HENECA (100 nM, 24 hours). D–E : MTT assay showing the effect of NaHS alone at 10–200 µM (D) or HENECA alone at 10–200 nM (E) on cell viability of SH-SY5Y cells. Aβ42 levels in conditioned media were measured by sandwich ELISA kit. Control values were adjusted to 100%. Data are given as means ± S.E.M, n = 6. ## p

    Techniques Used: Expressing, Concentration Assay, MTT Assay, Sandwich ELISA

    Schematic diagram showing the inhibitory effect of H 2 S on HENECA induced Aβ generation in SH-SY5Y cells. APP is an integral membrane protein which undergoes post-translational modification such as glycosylation during its transfer through intracellular secretory pathway. The mature isoform of APP (i.e. APP holoprotein) is then acted upon by β- and γ-secretases to generate Aβ. The A2A receptor agonist, HENECA, induces production of Aβ42 in SH-SY5Y cells via cAMP/PKA/CREB pathway. It enhances both synthesis and maturation processes of APP increasing total APP production. It also stimulates γ-secretase activity in mAPP cleavage resulting in Aβ generation. H 2 S not only interferes with the step of APP maturation, but also attenuates the production of APP holoprotein. By inhibiting AC (and subsequent cAMP production), H 2 S also inhibits γ-secretase activity. It ultimately leads to decreased production in Aβ.
    Figure Legend Snippet: Schematic diagram showing the inhibitory effect of H 2 S on HENECA induced Aβ generation in SH-SY5Y cells. APP is an integral membrane protein which undergoes post-translational modification such as glycosylation during its transfer through intracellular secretory pathway. The mature isoform of APP (i.e. APP holoprotein) is then acted upon by β- and γ-secretases to generate Aβ. The A2A receptor agonist, HENECA, induces production of Aβ42 in SH-SY5Y cells via cAMP/PKA/CREB pathway. It enhances both synthesis and maturation processes of APP increasing total APP production. It also stimulates γ-secretase activity in mAPP cleavage resulting in Aβ generation. H 2 S not only interferes with the step of APP maturation, but also attenuates the production of APP holoprotein. By inhibiting AC (and subsequent cAMP production), H 2 S also inhibits γ-secretase activity. It ultimately leads to decreased production in Aβ.

    Techniques Used: Modification, Activity Assay

    Effect of NaHS on expression of A2A receptors. A–B : Representative gels (A) and histogram (B) demonstrating the effect of pretreatment with NaHS (100 µM, 12 hours) did not attenuate the effects of HENECA (100 nM, 24 hours) on protein expression of A2A receptor. C : Effect of NaHS (100 µM) on production of Aβ42 in cells pre-treated with A2A receptor antagonist, ZM 241385 (50 nM). Control values were adjusted to 100%. Data are given as means ± S.E.M, n = 4–6. ## p
    Figure Legend Snippet: Effect of NaHS on expression of A2A receptors. A–B : Representative gels (A) and histogram (B) demonstrating the effect of pretreatment with NaHS (100 µM, 12 hours) did not attenuate the effects of HENECA (100 nM, 24 hours) on protein expression of A2A receptor. C : Effect of NaHS (100 µM) on production of Aβ42 in cells pre-treated with A2A receptor antagonist, ZM 241385 (50 nM). Control values were adjusted to 100%. Data are given as means ± S.E.M, n = 4–6. ## p

    Techniques Used: Expressing

    7) Product Images from "Elevated cellular cholesterol in Familial Alzheimer’s presenilin 1 mutation is associated with lipid raft localization of β-amyloid precursor protein"

    Article Title: Elevated cellular cholesterol in Familial Alzheimer’s presenilin 1 mutation is associated with lipid raft localization of β-amyloid precursor protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0210535

    Our current model for the effects of PS1 mutant on APP localization in lipid rafts and Aβ production. Elevated cholesterol in PS1 mutant increases lipid rafts structures altering APP distribution into lipid rafts, which produces more Aβ42 peptides.
    Figure Legend Snippet: Our current model for the effects of PS1 mutant on APP localization in lipid rafts and Aβ production. Elevated cholesterol in PS1 mutant increases lipid rafts structures altering APP distribution into lipid rafts, which produces more Aβ42 peptides.

    Techniques Used: Mutagenesis

    Tebuconazole decreased secreted Aβ42 levels in CHO PS1 ΔE9 cells but not in PS1 WT cells. CHO PS1 WT and ΔE9 cells were pre-treated with 0, 1, 5, or 10 μM tebuconazole for 48 h, and Aβ levels were measured from the conditioned media using ( a ) Aβ40 specific (n = 12) or ( b ) Aβ42 specific ELISA kit (n = 12). One-way ANOVA: *p
    Figure Legend Snippet: Tebuconazole decreased secreted Aβ42 levels in CHO PS1 ΔE9 cells but not in PS1 WT cells. CHO PS1 WT and ΔE9 cells were pre-treated with 0, 1, 5, or 10 μM tebuconazole for 48 h, and Aβ levels were measured from the conditioned media using ( a ) Aβ40 specific (n = 12) or ( b ) Aβ42 specific ELISA kit (n = 12). One-way ANOVA: *p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    8) Product Images from "URMC-099 facilitates amyloid-β clearance in a murine model of Alzheimer’s disease"

    Article Title: URMC-099 facilitates amyloid-β clearance in a murine model of Alzheimer’s disease

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-018-1172-y

    URMC-099 reduces extracellular Aβ40 and Aβ42 levels in the APP/PS1 brain. The levels of Aβ40 ( a ) and Aβ42 ( b ) in an extracellular-enriched fraction were measured by human Aβ40- and Aβ42-specific ELISAs. Data are presented as mean ± S.E.M. a,b p
    Figure Legend Snippet: URMC-099 reduces extracellular Aβ40 and Aβ42 levels in the APP/PS1 brain. The levels of Aβ40 ( a ) and Aβ42 ( b ) in an extracellular-enriched fraction were measured by human Aβ40- and Aβ42-specific ELISAs. Data are presented as mean ± S.E.M. a,b p

    Techniques Used:

    9) Product Images from "Phenotypic Assays For β-Amyloid In Mouse Embryonic Stem Cell-derived Neurons"

    Article Title: Phenotypic Assays For β-Amyloid In Mouse Embryonic Stem Cell-derived Neurons

    Journal: Chemistry & biology

    doi: 10.1016/j.chembiol.2013.06.005

    Modeling Aβ-associated Synaptic Abnormalities in ESNs (A and B) At DIV 21 mES derived neurons were treated for 24 hours with DMSO control (Ctrl) or Aβ42 oligomer, subsequently fixed and co-stained for synaptic proteins PSD-95 (green) and synpatophysin (S-physin) (red). Nuclei are labeled with DAPI (blue). PSD-95 and S-physin particles were analyzed using Image J software. (C-F) Quantification of PSD-95 positive particles and S-physin positive particles using Image J software particle analysis. (G) DIV 21 ESN were treated with control or Aβ42 oligomer, fixed, diolistically labeled and imaged with confocal microscopy. (H-J) Spine density, length and dendrite diameter were quantified using Image J software. (K) Phosphorylation of CREB was detected with a pCREB specific antibody after no stimulation (NS) or NMDA stimulation (ST) of cultures with or without Aβ42 oligomer (Aβ) pretreatment. (L) Quantification of CREB and pCREB using infrared quantitative Western blot imaging system with SEM shown by error bars.
    Figure Legend Snippet: Modeling Aβ-associated Synaptic Abnormalities in ESNs (A and B) At DIV 21 mES derived neurons were treated for 24 hours with DMSO control (Ctrl) or Aβ42 oligomer, subsequently fixed and co-stained for synaptic proteins PSD-95 (green) and synpatophysin (S-physin) (red). Nuclei are labeled with DAPI (blue). PSD-95 and S-physin particles were analyzed using Image J software. (C-F) Quantification of PSD-95 positive particles and S-physin positive particles using Image J software particle analysis. (G) DIV 21 ESN were treated with control or Aβ42 oligomer, fixed, diolistically labeled and imaged with confocal microscopy. (H-J) Spine density, length and dendrite diameter were quantified using Image J software. (K) Phosphorylation of CREB was detected with a pCREB specific antibody after no stimulation (NS) or NMDA stimulation (ST) of cultures with or without Aβ42 oligomer (Aβ) pretreatment. (L) Quantification of CREB and pCREB using infrared quantitative Western blot imaging system with SEM shown by error bars.

    Techniques Used: Derivative Assay, Staining, Labeling, Software, Confocal Microscopy, Western Blot, Imaging

    ESNs Harbor Machinery for APP Processing and Recapitulate Phenotype Associated with PS1 FAD (A) APP and BACE1 expression in mES cells and ESNs at DIV 7, 14 and 21. (B) Components of the γ-secretatse, PS1, APH-1, NCT and PEN2 detected by Western blot analysis of the cell lysates using indicated antibodies. (C) Gradient centrifugation shows APP and BACE1 are enriched in the late endosome compartment with Syn6 and Rab11 but not at the top of the gradient with EEA1. (D) Western blot analysis of protein lysates from mES cell derived neurons infected with Lentivirus harboring human APP sw expressing APP and proteolytic fragents. Secreted APP fragments, sAPPβ and sAPPα, were immunoprecipitated from conditioned media and APP and CTFs were detected in cell lysates. (E-F) Aβ40 and Aβ42 were detected in conditioned medium using human specific ELISA and values were normalized to protein lysate concentration. (G) Three different FAD mutants of PS1 (ΔE9, M146V, and L286V) were introduced into mES cells which were subsequently subjected to directed differentiation into pyramidal neurons. A representative Western blot shows transgene expression in the neurons derived from these clonal mES cell lines using an anti-human PS1 specific antibody (PS1-NT). (H) PS FAD-expressing cells undergo normal directed differentiation into pyramidal neurons indicated by expression of neuronal proteins. (I) ELISA detection of Aβ42 and Aβ40 from conditioned media of ESNs stably expressing PS-FAD mutants infected at DIV 7 or 14 with APP sw Lentiviral particles resulted in enhanced ratio of Aβ42/Aβ40. Lentiviral gene transfer did not affect the viability of ESNs (data not shown).
    Figure Legend Snippet: ESNs Harbor Machinery for APP Processing and Recapitulate Phenotype Associated with PS1 FAD (A) APP and BACE1 expression in mES cells and ESNs at DIV 7, 14 and 21. (B) Components of the γ-secretatse, PS1, APH-1, NCT and PEN2 detected by Western blot analysis of the cell lysates using indicated antibodies. (C) Gradient centrifugation shows APP and BACE1 are enriched in the late endosome compartment with Syn6 and Rab11 but not at the top of the gradient with EEA1. (D) Western blot analysis of protein lysates from mES cell derived neurons infected with Lentivirus harboring human APP sw expressing APP and proteolytic fragents. Secreted APP fragments, sAPPβ and sAPPα, were immunoprecipitated from conditioned media and APP and CTFs were detected in cell lysates. (E-F) Aβ40 and Aβ42 were detected in conditioned medium using human specific ELISA and values were normalized to protein lysate concentration. (G) Three different FAD mutants of PS1 (ΔE9, M146V, and L286V) were introduced into mES cells which were subsequently subjected to directed differentiation into pyramidal neurons. A representative Western blot shows transgene expression in the neurons derived from these clonal mES cell lines using an anti-human PS1 specific antibody (PS1-NT). (H) PS FAD-expressing cells undergo normal directed differentiation into pyramidal neurons indicated by expression of neuronal proteins. (I) ELISA detection of Aβ42 and Aβ40 from conditioned media of ESNs stably expressing PS-FAD mutants infected at DIV 7 or 14 with APP sw Lentiviral particles resulted in enhanced ratio of Aβ42/Aβ40. Lentiviral gene transfer did not affect the viability of ESNs (data not shown).

    Techniques Used: Expressing, Western Blot, Gradient Centrifugation, Derivative Assay, Infection, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Stable Transfection

    Analysis of mES Cells and ESNs Isolated From Tg2576 AD Mouse Model (A) Tg2576 mES-derived neurons identified by nuclear DAPI stain, express neuronal β-tubulinIII (TUJ-1) (green) and pyramidal cell protein EMX1 (red). Scale bar represents 40μm. (B) Western blot analysis of protein lysates from Tg2576 mES-derived neuronal culture harvested at indicated DIV express APP, cleaved APP fragments (APP-CTF) and neuronal proteins synaptophysin, CamKIIα and neuronal β-tubulinIII (TUJ-1). α-Actin is a loading control. (C) Cell lysates from DIV 7 Tg2576 ES-derived neurons treated with indicated compounds were subjected to Western blot detection with 6E10 (APP-FL), APP-CTmax (APP-CTF) and antibodies to indicated proteins. (D) Secreted sAPPα and APPβ were immunoprecipitated from conditioned medium after treatment of Tg2576 ES-derived neurons with the indicated compounds. (E, F) Secreted Aβ levels detected using human specific Aβ40 or 42 ELISA kit. Aβ40 and Aβ42 show characteristic sensitivity to BSI and GSI inhibition. IC 50 values for the BACE1 inhibitor were 229.7nM and 28.1nM for Aβ40 and Aβ42 respectively. IC 50 values for Compound E were 136.9pM and 102.2pM Aβ40 and Aβ42 respectively. The Z’ factors were 0.85 for Aβ40 and 0.5 for Aβ42 indicating an excellent assay for HTS.
    Figure Legend Snippet: Analysis of mES Cells and ESNs Isolated From Tg2576 AD Mouse Model (A) Tg2576 mES-derived neurons identified by nuclear DAPI stain, express neuronal β-tubulinIII (TUJ-1) (green) and pyramidal cell protein EMX1 (red). Scale bar represents 40μm. (B) Western blot analysis of protein lysates from Tg2576 mES-derived neuronal culture harvested at indicated DIV express APP, cleaved APP fragments (APP-CTF) and neuronal proteins synaptophysin, CamKIIα and neuronal β-tubulinIII (TUJ-1). α-Actin is a loading control. (C) Cell lysates from DIV 7 Tg2576 ES-derived neurons treated with indicated compounds were subjected to Western blot detection with 6E10 (APP-FL), APP-CTmax (APP-CTF) and antibodies to indicated proteins. (D) Secreted sAPPα and APPβ were immunoprecipitated from conditioned medium after treatment of Tg2576 ES-derived neurons with the indicated compounds. (E, F) Secreted Aβ levels detected using human specific Aβ40 or 42 ELISA kit. Aβ40 and Aβ42 show characteristic sensitivity to BSI and GSI inhibition. IC 50 values for the BACE1 inhibitor were 229.7nM and 28.1nM for Aβ40 and Aβ42 respectively. IC 50 values for Compound E were 136.9pM and 102.2pM Aβ40 and Aβ42 respectively. The Z’ factors were 0.85 for Aβ40 and 0.5 for Aβ42 indicating an excellent assay for HTS.

    Techniques Used: Isolation, Derivative Assay, Staining, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Inhibition

    10) Product Images from "Role of toll-like receptor signalling in Aβ uptake and clearance"

    Article Title: Role of toll-like receptor signalling in Aβ uptake and clearance

    Journal: Brain : a journal of neurology

    doi: 10.1093/brain/awl249

    Detection of ingested Aβ42 in BV-2 cells by fluorescent immunocytochemistry. After treatment with LPS (100 ng/ml) ( A ), PGN (13.3 μg/ml) ( B ) or CpG-ODN (0.51 μM) ( C ), ingested Aβ42 in BV-2 cells shows fluorescence by immunocytochemistry using 6E10 antibody and anti-mouse IgG antibody coupled with Alexa Fluor 488. BV-2 cells treated with control ODN ( D ) or PBS ( E ) show little to no fluorescence. Scale bars 250 μm.
    Figure Legend Snippet: Detection of ingested Aβ42 in BV-2 cells by fluorescent immunocytochemistry. After treatment with LPS (100 ng/ml) ( A ), PGN (13.3 μg/ml) ( B ) or CpG-ODN (0.51 μM) ( C ), ingested Aβ42 in BV-2 cells shows fluorescence by immunocytochemistry using 6E10 antibody and anti-mouse IgG antibody coupled with Alexa Fluor 488. BV-2 cells treated with control ODN ( D ) or PBS ( E ) show little to no fluorescence. Scale bars 250 μm.

    Techniques Used: Immunocytochemistry, Fluorescence

    Effects of inhibitors on Aβ42 uptake by LPS-activated BV-2 cells. After stimulation with LPS (100 ng/ml), BV-2 cells were treated with pertussis toxin (PTX) (100 or 500 ng/ml), anti-mouse CD14 antibody (20 μg/ml) or fucoidan (50 μg/ml) and incubated with oligomerized Aβ for 24 h. Ingested Aβ42 was detected by western blots using 6E10 antibody ( A ). Oligomerized Aβ42 was similarly analysed for verification ( A , the extreme right lane). The membranes were reprobed with GAPDH-specific antibody for normalization. The amounts of Aβ uptake by LPS-activated BV-2 cells after treatment with chemical inhibitors were compared with those by LPS-activated BV-2 cells without inhibitors by densitometric analysis of the western blots ( B ). * P
    Figure Legend Snippet: Effects of inhibitors on Aβ42 uptake by LPS-activated BV-2 cells. After stimulation with LPS (100 ng/ml), BV-2 cells were treated with pertussis toxin (PTX) (100 or 500 ng/ml), anti-mouse CD14 antibody (20 μg/ml) or fucoidan (50 μg/ml) and incubated with oligomerized Aβ for 24 h. Ingested Aβ42 was detected by western blots using 6E10 antibody ( A ). Oligomerized Aβ42 was similarly analysed for verification ( A , the extreme right lane). The membranes were reprobed with GAPDH-specific antibody for normalization. The amounts of Aβ uptake by LPS-activated BV-2 cells after treatment with chemical inhibitors were compared with those by LPS-activated BV-2 cells without inhibitors by densitometric analysis of the western blots ( B ). * P

    Techniques Used: Incubation, Western Blot

    The clearance of Aβ42 from culture media by activation of TLRs on microglia (BV-2 cells). BV-2 cells were treated with LPS at the concentrations of 1.2, 11 and 100 ng/ml, CpG oligodeoxynucleotides (CpG-ODN) at 0.06 and 0.51 μM or peptidoglycan (PGN) at 1.5 and 13.3 μg/ml. BV-2 cells treated with the TLR ligands were incubated with oligomerized Aβ42 for 24 h. As controls, BV-2 cells treated with Control ODN or PBS were used. The concentrations of residual Aβ42 in the culture media were determined by Aβ42-specific sandwich ELISA. The ratios of the Aβ42 concentrations in TLR ligand-treaded media to that in PBS-treated medium are shown. After stimulation with every TLR ligand tested, residual Aβ42 in the medium was reduced by ~50% at the TLR ligand concentrations tested, compared with that in the media from BV-2 cells treated with PBS or control ODN ( P
    Figure Legend Snippet: The clearance of Aβ42 from culture media by activation of TLRs on microglia (BV-2 cells). BV-2 cells were treated with LPS at the concentrations of 1.2, 11 and 100 ng/ml, CpG oligodeoxynucleotides (CpG-ODN) at 0.06 and 0.51 μM or peptidoglycan (PGN) at 1.5 and 13.3 μg/ml. BV-2 cells treated with the TLR ligands were incubated with oligomerized Aβ42 for 24 h. As controls, BV-2 cells treated with Control ODN or PBS were used. The concentrations of residual Aβ42 in the culture media were determined by Aβ42-specific sandwich ELISA. The ratios of the Aβ42 concentrations in TLR ligand-treaded media to that in PBS-treated medium are shown. After stimulation with every TLR ligand tested, residual Aβ42 in the medium was reduced by ~50% at the TLR ligand concentrations tested, compared with that in the media from BV-2 cells treated with PBS or control ODN ( P

    Techniques Used: Activation Assay, Incubation, Sandwich ELISA

    Quantification of buffer-soluble and insoluble Aβ in the cerebrum by the Aβ40- and Aβ42-specific ELISA. The cerebral buffer-soluble Aβ40 content in Tlr Lps-d / Tlr Lps-d mice is higher than that in TLR4 wild-type mice ( A ). The cerebral buffer-soluble Aβ42 content in Tlr Lps-d / Tlr Lps-d mice is higher than that in Tlr Lps-d / θ mice ( B ). The amount of total buffer-soluble Aβ (Aβ40 + Aβ42) is higher than that in TLR4 wild-type mice ( C ). The cerebral insoluble Aβ40 content in Tlr Lps-d / Tlr Lps-d mice is higher than that in Tlr Lps-d / θ mice ( D ). The cerebral insoluble Aβ42 content in Tlr Lps-d / Tlr Lps-d mice is higher than that in TLR4 wild-type mice ( E ). The amount of total insoluble Aβ (Aβ40 + Aβ42) in Tlr Lps-d / Tlr Lps-d mice is higher than that in TLR4 wild-type mice ( F ).
    Figure Legend Snippet: Quantification of buffer-soluble and insoluble Aβ in the cerebrum by the Aβ40- and Aβ42-specific ELISA. The cerebral buffer-soluble Aβ40 content in Tlr Lps-d / Tlr Lps-d mice is higher than that in TLR4 wild-type mice ( A ). The cerebral buffer-soluble Aβ42 content in Tlr Lps-d / Tlr Lps-d mice is higher than that in Tlr Lps-d / θ mice ( B ). The amount of total buffer-soluble Aβ (Aβ40 + Aβ42) is higher than that in TLR4 wild-type mice ( C ). The cerebral insoluble Aβ40 content in Tlr Lps-d / Tlr Lps-d mice is higher than that in Tlr Lps-d / θ mice ( D ). The cerebral insoluble Aβ42 content in Tlr Lps-d / Tlr Lps-d mice is higher than that in TLR4 wild-type mice ( E ). The amount of total insoluble Aβ (Aβ40 + Aβ42) in Tlr Lps-d / Tlr Lps-d mice is higher than that in TLR4 wild-type mice ( F ).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay

    Detection of Aβ42 ingested by BV-2 cells after stimulation with TLR ligands. After activation of microglia with TLR ligands, ingested Aβ42 by BV-2 cells are detected by western blots using 6E10 antibody. To monitor the degree of Aβ oligomerization for each experiment, oligomerized Aβ42 was similarly analyzed (the extremely right lanes). Intracellular monomer and aggregates of Aβ are readily detectable in the cell lysates from BV-2 cells treated with TLR ligands at the concentrations of 1.2, 3.7, 11 and 33 ng/ml for LPS ( A ), 0.02, 0.06, 0.18 and 0.54 μM for CpG-ODN ( B ) and 0.5, 1.5, 4.5 and 40 μg/ml for PGN ( C ), while Aβ is barely visible in the cell lysate from BV-2 cells without treatment with TLR ligands. The membranes were reprobed with GAPDH-specific antibody for normalization.
    Figure Legend Snippet: Detection of Aβ42 ingested by BV-2 cells after stimulation with TLR ligands. After activation of microglia with TLR ligands, ingested Aβ42 by BV-2 cells are detected by western blots using 6E10 antibody. To monitor the degree of Aβ oligomerization for each experiment, oligomerized Aβ42 was similarly analyzed (the extremely right lanes). Intracellular monomer and aggregates of Aβ are readily detectable in the cell lysates from BV-2 cells treated with TLR ligands at the concentrations of 1.2, 3.7, 11 and 33 ng/ml for LPS ( A ), 0.02, 0.06, 0.18 and 0.54 μM for CpG-ODN ( B ) and 0.5, 1.5, 4.5 and 40 μg/ml for PGN ( C ), while Aβ is barely visible in the cell lysate from BV-2 cells without treatment with TLR ligands. The membranes were reprobed with GAPDH-specific antibody for normalization.

    Techniques Used: Activation Assay, Western Blot

    Detection of ingested Aβ42 in primary TLR4 wild-type and Tlr Lps-d / Tlr Lps-d microglia by fluorescent immunocytochemistry. Primary TLR4 wild-type ( A–C ) and Tlr Lps-d / Tlr Lps-d ( D – F ) microglia were treated with LPS (100 ng/ml) ( A and D ), CpG-ODN (0.51 μM) ( B and E ) or PBS ( C and F ) and exposed to oligomerized Aβ (0.25 μM) for 24 h. Ingested Aβ42 in the cells shows fluorescence by immunocytochemistry using 6E10 antibody and anti-mouse IgG antibody coupled with Alexa Fluor 488. Arrows indicate ingested Aβ. Scale bars 40 μm.
    Figure Legend Snippet: Detection of ingested Aβ42 in primary TLR4 wild-type and Tlr Lps-d / Tlr Lps-d microglia by fluorescent immunocytochemistry. Primary TLR4 wild-type ( A–C ) and Tlr Lps-d / Tlr Lps-d ( D – F ) microglia were treated with LPS (100 ng/ml) ( A and D ), CpG-ODN (0.51 μM) ( B and E ) or PBS ( C and F ) and exposed to oligomerized Aβ (0.25 μM) for 24 h. Ingested Aβ42 in the cells shows fluorescence by immunocytochemistry using 6E10 antibody and anti-mouse IgG antibody coupled with Alexa Fluor 488. Arrows indicate ingested Aβ. Scale bars 40 μm.

    Techniques Used: Immunocytochemistry, Fluorescence

    11) Product Images from "Palmitate induces transcriptional regulation of BACE1 and presenilin by STAT3 in neurons mediated by astrocytes"

    Article Title: Palmitate induces transcriptional regulation of BACE1 and presenilin by STAT3 in neurons mediated by astrocytes

    Journal: Experimental neurology

    doi: 10.1016/j.expneurol.2013.08.004

    Aβ42 fold change
    Figure Legend Snippet: Aβ42 fold change

    Techniques Used:

    12) Product Images from "Effects of Secondary Metabolite Extract from Phomopsis occulta on β-Amyloid Aggregation"

    Article Title: Effects of Secondary Metabolite Extract from Phomopsis occulta on β-Amyloid Aggregation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0109438

    Screening of bioactive fractions from Ph. occulta secondary metabolite extracts using an E. coli cell model. A: Inhibitory effect on Aβ42 aggregation of Ph. occulta secondary metabolite extracts. ME: mycelia extracts; BE: broth extracts; 0, 1, 2 and 3 refer to molar salt concentrations in the cultures. B: Inhibitory effect on Aβ42 aggregation of ME0 fractions. ME0-W-F1 to ME0-W-F5 are water soluble fractions separated by column chromatography using Diaion-20 resin and a water/methanol mobile phase. Fraction concentrations were 200 µg/ml in each case; EGCG was used as positive control (100 µg/ml). Values represent mean of means ± SD of four separate experiments, each performed in triplicate.
    Figure Legend Snippet: Screening of bioactive fractions from Ph. occulta secondary metabolite extracts using an E. coli cell model. A: Inhibitory effect on Aβ42 aggregation of Ph. occulta secondary metabolite extracts. ME: mycelia extracts; BE: broth extracts; 0, 1, 2 and 3 refer to molar salt concentrations in the cultures. B: Inhibitory effect on Aβ42 aggregation of ME0 fractions. ME0-W-F1 to ME0-W-F5 are water soluble fractions separated by column chromatography using Diaion-20 resin and a water/methanol mobile phase. Fraction concentrations were 200 µg/ml in each case; EGCG was used as positive control (100 µg/ml). Values represent mean of means ± SD of four separate experiments, each performed in triplicate.

    Techniques Used: Column Chromatography, Positive Control

    ME0-W-F1 reduces aggregation of Aβ42-EGFP and the early onset familial mutation Aβ42E22G-mCherry). (A, B) HEK293 transiently transfected with pcDNA3-Aβ42–EGFP (grey bars) and the Aβ42E22G-mCherry construct pATNRW20 (black bars) and treated with ME0-W-F1 at 17.5 µg/ml (H) and 1.75 µg/ml (L), a positive control (10 µM EGCG) and a negative control (DMSO only). For each construct and treatment, three fields of approximately 200 cells (i.e. n = 200 for each field) were counted for aggregates and odds ratios calculated. Error bars indicate 95% confidence interval for the odds ratio. Treatments of the Aβ42E22G-mCherry transfections with ME0-W-F1 (H and L) and EGCG were all statistically significant with a probability of P
    Figure Legend Snippet: ME0-W-F1 reduces aggregation of Aβ42-EGFP and the early onset familial mutation Aβ42E22G-mCherry). (A, B) HEK293 transiently transfected with pcDNA3-Aβ42–EGFP (grey bars) and the Aβ42E22G-mCherry construct pATNRW20 (black bars) and treated with ME0-W-F1 at 17.5 µg/ml (H) and 1.75 µg/ml (L), a positive control (10 µM EGCG) and a negative control (DMSO only). For each construct and treatment, three fields of approximately 200 cells (i.e. n = 200 for each field) were counted for aggregates and odds ratios calculated. Error bars indicate 95% confidence interval for the odds ratio. Treatments of the Aβ42E22G-mCherry transfections with ME0-W-F1 (H and L) and EGCG were all statistically significant with a probability of P

    Techniques Used: Mutagenesis, Transfection, Construct, Positive Control, Negative Control

    Effect of ME0-W-F1 on aggregation of Aβ42 analysed by SDS-PAGE and quantitative analysis with Quantitative One. Low (Aβ42: ME0-W-F1 = 1∶1) and high (Aβ42: ME0-W-F1 = 1∶4) concentrations of ME0-W-F1 were used. Values represent mean ± SD of three replicates. The data were evaluated by t -test, * p
    Figure Legend Snippet: Effect of ME0-W-F1 on aggregation of Aβ42 analysed by SDS-PAGE and quantitative analysis with Quantitative One. Low (Aβ42: ME0-W-F1 = 1∶1) and high (Aβ42: ME0-W-F1 = 1∶4) concentrations of ME0-W-F1 were used. Values represent mean ± SD of three replicates. The data were evaluated by t -test, * p

    Techniques Used: SDS Page

    Inhibitory effects of ME0-W-F1 on the transformation of secondary structure of Aβ42 by FT-IR. A: Aβ42 alone; B: Aβ42 with ME0-W-F1; C: change in β-sheet content during incubation with (+) or without (−) ME0-W-F1. Time: 0, 0.5, 1, 2 and 4 days. Values represent mean ± SD of three separate experiments.
    Figure Legend Snippet: Inhibitory effects of ME0-W-F1 on the transformation of secondary structure of Aβ42 by FT-IR. A: Aβ42 alone; B: Aβ42 with ME0-W-F1; C: change in β-sheet content during incubation with (+) or without (−) ME0-W-F1. Time: 0, 0.5, 1, 2 and 4 days. Values represent mean ± SD of three separate experiments.

    Techniques Used: Transformation Assay, Incubation

    Protective effect of ME0-W-F1 in SH-SY5Y cells against cytotoxicity induced by aggregation of Aβ42 (10 µM), as shown by MTT analysis. Four concentrations (µg/ml) were used, with EGCG as positive control. Values represent mean of means ± SD of four separate experiments, each performed in triplicate (i.e. n = 12). The data were evaluated by two-way analysis of variance (ANOVA) followed by a post hoc test. *, **, ***, statistically significant from each other, p
    Figure Legend Snippet: Protective effect of ME0-W-F1 in SH-SY5Y cells against cytotoxicity induced by aggregation of Aβ42 (10 µM), as shown by MTT analysis. Four concentrations (µg/ml) were used, with EGCG as positive control. Values represent mean of means ± SD of four separate experiments, each performed in triplicate (i.e. n = 12). The data were evaluated by two-way analysis of variance (ANOVA) followed by a post hoc test. *, **, ***, statistically significant from each other, p

    Techniques Used: MTT Assay, Positive Control

    13) Product Images from "Disruption of amyloid precursor protein ubiquitination selectively increases amyloid β (Aβ) 40 levels via presenilin 2-mediated cleavage"

    Article Title: Disruption of amyloid precursor protein ubiquitination selectively increases amyloid β (Aβ) 40 levels via presenilin 2-mediated cleavage

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.818138

    APP 5R does not accumulate on cell surface, and γ-secretase cleavage does not produce more Aβ40 from APP 5R in a cell-free assay. A, representative Western blot of total and surface APP GFP from N2a cells expressing APP WT GFP or APP 5R GFP with quantification of Western blot, expressed as a fraction of surface to total APP GFP . Values are expressed in arbitrary units ( A.U. ) relative to APP WT GFP and denote mean ± S.E., n = 5 biological replicates. N.S. denotes no significance between samples means, as measured by Student's t test. B, levels of Aβ40 and Aβ42 from in vitro γ-secretase assay. Purified APP C99 substrate is incubated with purified γ-secretase membranes, and resultant Aβ levels are measured via ELISA. Aβ levels are expressed in arbitrary units ( A.U. ) relative to C99 WT . Values denote mean ± S.E., n = 6 biological replicates. No significance between samples means, as measured by Student's t test.
    Figure Legend Snippet: APP 5R does not accumulate on cell surface, and γ-secretase cleavage does not produce more Aβ40 from APP 5R in a cell-free assay. A, representative Western blot of total and surface APP GFP from N2a cells expressing APP WT GFP or APP 5R GFP with quantification of Western blot, expressed as a fraction of surface to total APP GFP . Values are expressed in arbitrary units ( A.U. ) relative to APP WT GFP and denote mean ± S.E., n = 5 biological replicates. N.S. denotes no significance between samples means, as measured by Student's t test. B, levels of Aβ40 and Aβ42 from in vitro γ-secretase assay. Purified APP C99 substrate is incubated with purified γ-secretase membranes, and resultant Aβ levels are measured via ELISA. Aβ levels are expressed in arbitrary units ( A.U. ) relative to C99 WT . Values denote mean ± S.E., n = 6 biological replicates. No significance between samples means, as measured by Student's t test.

    Techniques Used: Cell-Free Assay, Western Blot, Expressing, In Vitro, Purification, Incubation, Enzyme-linked Immunosorbent Assay

    Chemically induced cross-linking of APP and ubiquitin decreases Aβ40 levels. A, diagram describing the dimerization of DmrA and DmrC domains fused to APP and ubiquitin, respectively, after addition of A/C heterodimerizer to culture media. B, representative Western blot of N2a cells expressing APP DmrA-RFP alone or co-expressing APP DmrA-RFP and Ub DmrC were treated for 12 h with control solute ethanol or A/C heterodimerizer. C, cell culture media from B was measured for levels of secreted Aβ40 and Aβ42 measured via ELISA, and values are expressed in arbitrary units ( A.U. ) relative to APP WT DmrA-RFP alone. Values denote mean ± S.E., n = 3–6 biological replicates. *, p
    Figure Legend Snippet: Chemically induced cross-linking of APP and ubiquitin decreases Aβ40 levels. A, diagram describing the dimerization of DmrA and DmrC domains fused to APP and ubiquitin, respectively, after addition of A/C heterodimerizer to culture media. B, representative Western blot of N2a cells expressing APP DmrA-RFP alone or co-expressing APP DmrA-RFP and Ub DmrC were treated for 12 h with control solute ethanol or A/C heterodimerizer. C, cell culture media from B was measured for levels of secreted Aβ40 and Aβ42 measured via ELISA, and values are expressed in arbitrary units ( A.U. ) relative to APP WT DmrA-RFP alone. Values denote mean ± S.E., n = 3–6 biological replicates. *, p

    Techniques Used: Western Blot, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    Metabolism of APP Lys → Arg mutants is altered in N2a cells. A, representative Western blot of full-length APP GFP (FL-APP GFP ) and actin from cell lysate and sAPPα from cell media of N2a cells expressing APP GFP lysine-to-arginine mutants. Numbered lanes indicate APP lysine residue(s) mutated to arginine (WT = wild type; 3R = APP K724R/K725R/K726R; 5R = all C-terminal lysines mutated). B, quantification of FL-APP GFP and sAPPα (soluble N-terminal fragment from α-secretase cleavage) from Western blot, and quantification of Aβ40, Aβ42, and sAPPβ (soluble N-terminal fragment from β-secretase cleavage) by ELISA of cell culture media. FL-APP GFP is normalized to actin loading control and sAPPα, sAPPβ, Aβ40, and Aβ42 are normalized to FL-APP GFP . All are expressed in arbitrary units ( A.U. ) relative to APP WT GFP . Values denote mean ± S.E., n = 5–7 biological replicates. *, p
    Figure Legend Snippet: Metabolism of APP Lys → Arg mutants is altered in N2a cells. A, representative Western blot of full-length APP GFP (FL-APP GFP ) and actin from cell lysate and sAPPα from cell media of N2a cells expressing APP GFP lysine-to-arginine mutants. Numbered lanes indicate APP lysine residue(s) mutated to arginine (WT = wild type; 3R = APP K724R/K725R/K726R; 5R = all C-terminal lysines mutated). B, quantification of FL-APP GFP and sAPPα (soluble N-terminal fragment from α-secretase cleavage) from Western blot, and quantification of Aβ40, Aβ42, and sAPPβ (soluble N-terminal fragment from β-secretase cleavage) by ELISA of cell culture media. FL-APP GFP is normalized to actin loading control and sAPPα, sAPPβ, Aβ40, and Aβ42 are normalized to FL-APP GFP . All are expressed in arbitrary units ( A.U. ) relative to APP WT GFP . Values denote mean ± S.E., n = 5–7 biological replicates. *, p

    Techniques Used: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture

    Exosomal content of APP-CTF is reduced in N2a cells expressing APP 5R GFP . A, representative Western blot of full-length APP (FL-APP, 22C11 N-terminal marker), APP C-terminal fragment (APP-CTF GFP , CT-20 C-terminal marker), and Alix in the cell lysate and exosomes purified by ultracentrifugation of cell media from N2a cells expressing APP GFP . Bands corresponding to exogenous APP GFP , endogenous murine APP, and exogenous APP-CTF GFP and APP intracellular domain (AICD GFP ) are indicated by arrows. B, quantification of APP-CTF GFP in exosomes isolated from cell culture media of N2a cells expressing APP GFP . APP-CTF GFP levels are normalized to Alix, a marker of exosomes, and expressed in arbitrary units ( A.U. ) relative to APP WT GFP . Values denote mean ± S.E., n = 5 biological replicates. C, Aβ40 and Aβ42 associated with exosomes isolated from cell culture media of N2a cells expressing APP GFP as measured by ELISA. Values are expressed in arbitrary units ( A.U. ) relative to APP WT GFP . Values denote mean ± S.E., n = 4 biological replicates. **, p
    Figure Legend Snippet: Exosomal content of APP-CTF is reduced in N2a cells expressing APP 5R GFP . A, representative Western blot of full-length APP (FL-APP, 22C11 N-terminal marker), APP C-terminal fragment (APP-CTF GFP , CT-20 C-terminal marker), and Alix in the cell lysate and exosomes purified by ultracentrifugation of cell media from N2a cells expressing APP GFP . Bands corresponding to exogenous APP GFP , endogenous murine APP, and exogenous APP-CTF GFP and APP intracellular domain (AICD GFP ) are indicated by arrows. B, quantification of APP-CTF GFP in exosomes isolated from cell culture media of N2a cells expressing APP GFP . APP-CTF GFP levels are normalized to Alix, a marker of exosomes, and expressed in arbitrary units ( A.U. ) relative to APP WT GFP . Values denote mean ± S.E., n = 5 biological replicates. C, Aβ40 and Aβ42 associated with exosomes isolated from cell culture media of N2a cells expressing APP GFP as measured by ELISA. Values are expressed in arbitrary units ( A.U. ) relative to APP WT GFP . Values denote mean ± S.E., n = 4 biological replicates. **, p

    Techniques Used: Expressing, Western Blot, Marker, Purification, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

    14) Product Images from "Hypoxia Due to Cardiac Arrest Induces a Time-Dependent Increase in Serum Amyloid ? Levels in Humans"

    Article Title: Hypoxia Due to Cardiac Arrest Induces a Time-Dependent Increase in Serum Amyloid ? Levels in Humans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028263

    Features of Aβ42 elevation profiles were compared with 6-month overall cerebral outcome. ( A ) Magnitude of Aβ42 rise; ( B ) ratio of maximum to baseline Aβ42; ( C ) duration of major Aβ42 rise; ( D ) sum of A to C plus maximum slope of Aβ42 rise. Error bars are the standard error of the means.
    Figure Legend Snippet: Features of Aβ42 elevation profiles were compared with 6-month overall cerebral outcome. ( A ) Magnitude of Aβ42 rise; ( B ) ratio of maximum to baseline Aβ42; ( C ) duration of major Aβ42 rise; ( D ) sum of A to C plus maximum slope of Aβ42 rise. Error bars are the standard error of the means.

    Techniques Used:

    Assay characteristics. Arrays of femtoliter-volume wells permit isolation of 2.7 mm capture beads from a standard bead-based ELISA ( A ), enabling exquisite sensitivity to enzyme label by preventing fluorescent product from diffusing away into a bulk solution. At very low concentrations of Aβ42, beads contain either a single labeled immunocomplex or no complexes, giving rise to digital signal output corresponding to single molecules ( B ). Simultaneous counting of active wells across an array statistically powers estimates of average enzymes/bead. ( C ) Dose-response of digital immunoassay for Aβ42 (n = 3). Y-axis refers to average number of enzyme labels per individual microbead captured in the array. The concentration of label is reduced relative to standard immunoassays, resulting in improved signal∶background at very low Aβ42 concentration. Assay calibrators were purified Aβ42 (Merck) in PBS/BSA. (C inset) Limit of quantification (LoQ) was estimated from total coefficients of variation (CV) from five low panel members (spiked PBS/BSA panels, grey circles, and immunodepleted plasma, black circles) assayed repeatedly across five days. The Aβ42 concentration at which total assay imprecision reached 20% (LoQ) was 0.032 pg/mL. ( D ) Recovery at extremely low Aβ42 concentrations was tested by diluting spiked immunodepleted plasma with PBS/BSA zero calibrator (n = 3). Because samples are pre-diluted 4-fold prior to assay to reduce matrix effects, dilutions include an initial 4-fold dilution.
    Figure Legend Snippet: Assay characteristics. Arrays of femtoliter-volume wells permit isolation of 2.7 mm capture beads from a standard bead-based ELISA ( A ), enabling exquisite sensitivity to enzyme label by preventing fluorescent product from diffusing away into a bulk solution. At very low concentrations of Aβ42, beads contain either a single labeled immunocomplex or no complexes, giving rise to digital signal output corresponding to single molecules ( B ). Simultaneous counting of active wells across an array statistically powers estimates of average enzymes/bead. ( C ) Dose-response of digital immunoassay for Aβ42 (n = 3). Y-axis refers to average number of enzyme labels per individual microbead captured in the array. The concentration of label is reduced relative to standard immunoassays, resulting in improved signal∶background at very low Aβ42 concentration. Assay calibrators were purified Aβ42 (Merck) in PBS/BSA. (C inset) Limit of quantification (LoQ) was estimated from total coefficients of variation (CV) from five low panel members (spiked PBS/BSA panels, grey circles, and immunodepleted plasma, black circles) assayed repeatedly across five days. The Aβ42 concentration at which total assay imprecision reached 20% (LoQ) was 0.032 pg/mL. ( D ) Recovery at extremely low Aβ42 concentrations was tested by diluting spiked immunodepleted plasma with PBS/BSA zero calibrator (n = 3). Because samples are pre-diluted 4-fold prior to assay to reduce matrix effects, dilutions include an initial 4-fold dilution.

    Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay, Labeling, Concentration Assay, Purification

    Serum Aβ42 following resuscitation from cardiac arrest. CPC scores depicted are after discharge from the ICU and 6 months later. Panels on left ( A–C ) are profiles from patients exhibiting good outcomes, panels on the right ( D–F ) are from patients with poor outcome. ( E ) Illustration of Aβ42 profile analysis. Baseline Aβ42 was defined as the mean of the two lowest values in the initial 12 hours. The time of initial elevation was defined as the intersection between the baseline Aβ42 and the line of maximum ascension of the major elevation peak. The duration of the Aβ42 increase was defined as the difference between time of initial elevation and time beyond which no significant further rise was observed. The magnitude and maximum slope of rise are also indicated. ( A ) Patient exhibiting smallest relative increase in Aβ42 among all patients. For confirmation, the sample set was re-assayed on a different day. ( D ) Patient with poor outcome exhibiting the largest relative increase from baseline. ( C and F ) Patients with similar baseline Aβ42 and poor cerebral outcome upon discharge from the ICU. Six months later, patient BE had recovered good cerebral function, while patient LP had not. Error bars: standard deviation of triplicate measurements.
    Figure Legend Snippet: Serum Aβ42 following resuscitation from cardiac arrest. CPC scores depicted are after discharge from the ICU and 6 months later. Panels on left ( A–C ) are profiles from patients exhibiting good outcomes, panels on the right ( D–F ) are from patients with poor outcome. ( E ) Illustration of Aβ42 profile analysis. Baseline Aβ42 was defined as the mean of the two lowest values in the initial 12 hours. The time of initial elevation was defined as the intersection between the baseline Aβ42 and the line of maximum ascension of the major elevation peak. The duration of the Aβ42 increase was defined as the difference between time of initial elevation and time beyond which no significant further rise was observed. The magnitude and maximum slope of rise are also indicated. ( A ) Patient exhibiting smallest relative increase in Aβ42 among all patients. For confirmation, the sample set was re-assayed on a different day. ( D ) Patient with poor outcome exhibiting the largest relative increase from baseline. ( C and F ) Patients with similar baseline Aβ42 and poor cerebral outcome upon discharge from the ICU. Six months later, patient BE had recovered good cerebral function, while patient LP had not. Error bars: standard deviation of triplicate measurements.

    Techniques Used: Standard Deviation

    15) Product Images from "Preparation of pure populations of covalently stabilized amyloid β-protein oligomers of specific sizes"

    Article Title: Preparation of pure populations of covalently stabilized amyloid β-protein oligomers of specific sizes

    Journal: Analytical biochemistry

    doi: 10.1016/j.ab.2016.10.026

    Toxicity of wild type Aβ42 and [F10, Y42]Aβ42 LDH assays were performed on NGF-differentiated PC12 cells incubated for 48 h with freshly prepared, non-cross-linked (nXL) peptides, cross-linked (XL) peptides, or fibrils. Percent toxicity T = ((F Aβ – F medium )/(F full kill – F medium )) × 100, where F Aβ , F medium , and F full kill were LDH activities from Aβ containing samples, negative control (buffer volume equivalent with medium), and 1 μM staurosporine alone, respectively.
    Figure Legend Snippet: Toxicity of wild type Aβ42 and [F10, Y42]Aβ42 LDH assays were performed on NGF-differentiated PC12 cells incubated for 48 h with freshly prepared, non-cross-linked (nXL) peptides, cross-linked (XL) peptides, or fibrils. Percent toxicity T = ((F Aβ – F medium )/(F full kill – F medium )) × 100, where F Aβ , F medium , and F full kill were LDH activities from Aβ containing samples, negative control (buffer volume equivalent with medium), and 1 μM staurosporine alone, respectively.

    Techniques Used: Incubation, Negative Control

    Effects of DMSO and urea on Aβ oligomer distributions (a) 12.5, 25, and 50% DMSO were added to cross-linked [F10,Y42]Aβ42 after which SDS-PAGE and silver staining were performed. Non-cross-linked (nXL) and cross-linked (XL) Aβ42 and [F10,Y42]Aβ42 were analyzed, with or without DMSO pre-treatment, by (b) SDS-PAGE or (c) SDS-PAGE with 6 M urea. Within each of the images (a-c), all lanes were from the same gel. However, to make comparisons among treatments easier, the lanes have been reordered. The oligomer order noted on the right of each gel corresponds to the cross-linked oligomer size. (d) Intensity profiles of cross-linked [F10, Y42]Aβ42 with and without 25% DMSO. (e) Intensity profiles of cross-linked [F10, Y42]Aβ42, with and without 25% DMSO, electrophoresed in the presence of 6M urea.
    Figure Legend Snippet: Effects of DMSO and urea on Aβ oligomer distributions (a) 12.5, 25, and 50% DMSO were added to cross-linked [F10,Y42]Aβ42 after which SDS-PAGE and silver staining were performed. Non-cross-linked (nXL) and cross-linked (XL) Aβ42 and [F10,Y42]Aβ42 were analyzed, with or without DMSO pre-treatment, by (b) SDS-PAGE or (c) SDS-PAGE with 6 M urea. Within each of the images (a-c), all lanes were from the same gel. However, to make comparisons among treatments easier, the lanes have been reordered. The oligomer order noted on the right of each gel corresponds to the cross-linked oligomer size. (d) Intensity profiles of cross-linked [F10, Y42]Aβ42 with and without 25% DMSO. (e) Intensity profiles of cross-linked [F10, Y42]Aβ42, with and without 25% DMSO, electrophoresed in the presence of 6M urea.

    Techniques Used: SDS Page, Silver Staining

    Purity of oligomers at each step of the purification procedure (a) Cross-linked [F10,Y42]Aβ42 was treated with 25% DMSO, subjected to SDS-PAGE, and the gel was stained using the zinc/imidazole method. The zinc/imidazole is a negative stain, thus the protein bands remain translucent upon visualization. (b) Oligomer bands were excised and electrophoresed on a second SDS-PAGE, containing 6 M urea, after which the gel was stained using zinc/ imidazole. Numbers below the images correspond to oligomer order (i.e., 2 is dimer, 3 is trimer, etc.). (c) Oligomer bands were excised from the urea containing gel and subjected to electro-elution. The isolated oligomers then were characterized by SDS-PAGE and silver staining.
    Figure Legend Snippet: Purity of oligomers at each step of the purification procedure (a) Cross-linked [F10,Y42]Aβ42 was treated with 25% DMSO, subjected to SDS-PAGE, and the gel was stained using the zinc/imidazole method. The zinc/imidazole is a negative stain, thus the protein bands remain translucent upon visualization. (b) Oligomer bands were excised and electrophoresed on a second SDS-PAGE, containing 6 M urea, after which the gel was stained using zinc/ imidazole. Numbers below the images correspond to oligomer order (i.e., 2 is dimer, 3 is trimer, etc.). (c) Oligomer bands were excised from the urea containing gel and subjected to electro-elution. The isolated oligomers then were characterized by SDS-PAGE and silver staining.

    Techniques Used: Purification, SDS Page, Staining, Isolation, Silver Staining

    Stability of cross-linked Aβ42 oligomers Cross-linked (a) Aβ42 and (b) [F10,Y42]Aβ42 oligomers were electrophoresed by SDS-PAGE and excised. These excised oligomer bands were examined by SDS-PAGE and silver stained. Open arrowheads in images (a) and (b) denote the positions where the nominal pure oligomer band should be in each lane. (Reprinted with permission from Yamin et al. ]. Copyright 2015 American Chemical Society.) (c) Oligomer bands of [F10,Y42]Aβ42 (corresponding to oligomers shown in panel (b) were excised and their component oligomers were isolated using electro-elution. Subsequent SDS-PAGE and silver staining reveals oligomer purity. The vertical white line between lanes labeled 2 and 3 in panel (c) comes from the deletion of the image of a lane that was blank in the original gel.
    Figure Legend Snippet: Stability of cross-linked Aβ42 oligomers Cross-linked (a) Aβ42 and (b) [F10,Y42]Aβ42 oligomers were electrophoresed by SDS-PAGE and excised. These excised oligomer bands were examined by SDS-PAGE and silver stained. Open arrowheads in images (a) and (b) denote the positions where the nominal pure oligomer band should be in each lane. (Reprinted with permission from Yamin et al. ]. Copyright 2015 American Chemical Society.) (c) Oligomer bands of [F10,Y42]Aβ42 (corresponding to oligomers shown in panel (b) were excised and their component oligomers were isolated using electro-elution. Subsequent SDS-PAGE and silver staining reveals oligomer purity. The vertical white line between lanes labeled 2 and 3 in panel (c) comes from the deletion of the image of a lane that was blank in the original gel.

    Techniques Used: SDS Page, Staining, Isolation, Silver Staining, Labeling

    16) Product Images from "Triterpenoid CDDO-methylamide improves memory and decreases amyloid plaques in a transgenic mouse model of Alzheimer's disease"

    Article Title: Triterpenoid CDDO-methylamide improves memory and decreases amyloid plaques in a transgenic mouse model of Alzheimer's disease

    Journal: Journal of neurochemistry

    doi: 10.1111/j.1471-4159.2009.05970.x

    Cyano-3,12-dioxooleana-1,9-dien-28-oic acid methylamide (CDDO-MA) decreased levels of sodium dodecyl sulfate-soluble Aβ42 but did not affect amyloid precursor protein (APP) processing. Levels of Aβ42 (a) and Aβ40 (b) by ELISA. Administration of CDDO-MA reduced significantly levels of Aβ42 in Tg19959 mice (a; * p = 0.0453). Levels of full length APP (c), α-CTFs (d), β-CTFs (e), neprilysin (f) and insulin degrading enzyme (g) in Tg19959 mice were measured by western blots. Data were expressed as ratios to tubulin (means ± SE). No differences were found between Tg19959 mice fed CDDO-MA and Tg19959 mice fed control (CON) chow. ns, not significant; CTF, carboxyterminal fragment of APP.
    Figure Legend Snippet: Cyano-3,12-dioxooleana-1,9-dien-28-oic acid methylamide (CDDO-MA) decreased levels of sodium dodecyl sulfate-soluble Aβ42 but did not affect amyloid precursor protein (APP) processing. Levels of Aβ42 (a) and Aβ40 (b) by ELISA. Administration of CDDO-MA reduced significantly levels of Aβ42 in Tg19959 mice (a; * p = 0.0453). Levels of full length APP (c), α-CTFs (d), β-CTFs (e), neprilysin (f) and insulin degrading enzyme (g) in Tg19959 mice were measured by western blots. Data were expressed as ratios to tubulin (means ± SE). No differences were found between Tg19959 mice fed CDDO-MA and Tg19959 mice fed control (CON) chow. ns, not significant; CTF, carboxyterminal fragment of APP.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Western Blot

    Cyano-3,12-dioxooleana-1,9-dien-28-oic acid methylamide (CDDO-MA) decreased plaque burden and microglia in the hippocampus of Tg19959 mice. (a) Representative photograph of plaques detected by Aβ42 antibody, AB5078P. (b) Plaque count and (c) percent area occupied by plaque in Tg19959 mice fed CDDO-MA or control (CON) chow (means ± SE). In hippocampus, CDDO-MA reduced significantly the plaque count (b; n = 6 per group; * p = 0.0211) and the percent area occupied by plaque (c; n = 6 per group; * p = 0.0072). (d) Representative photographs of microglial staining detected by CD40 antibody with high magnification views in the insets. (e) Area covered by microglia (means ± SE). In hippocampus, CDDO-MA reduced the area covered by microglia ( n = 6 per group; * p = 0.0437). ns, not significant.
    Figure Legend Snippet: Cyano-3,12-dioxooleana-1,9-dien-28-oic acid methylamide (CDDO-MA) decreased plaque burden and microglia in the hippocampus of Tg19959 mice. (a) Representative photograph of plaques detected by Aβ42 antibody, AB5078P. (b) Plaque count and (c) percent area occupied by plaque in Tg19959 mice fed CDDO-MA or control (CON) chow (means ± SE). In hippocampus, CDDO-MA reduced significantly the plaque count (b; n = 6 per group; * p = 0.0211) and the percent area occupied by plaque (c; n = 6 per group; * p = 0.0072). (d) Representative photographs of microglial staining detected by CD40 antibody with high magnification views in the insets. (e) Area covered by microglia (means ± SE). In hippocampus, CDDO-MA reduced the area covered by microglia ( n = 6 per group; * p = 0.0437). ns, not significant.

    Techniques Used: Mouse Assay, Staining

    17) Product Images from "RETROMER DISRUPTION PROMOTES AMYLOIDOGENIC APP PROCESSING *"

    Article Title: RETROMER DISRUPTION PROMOTES AMYLOIDOGENIC APP PROCESSING *

    Journal: Neurobiology of disease

    doi: 10.1016/j.nbd.2011.04.002

    Aβ40 secretion is decreased and the Aβ42:Aβ40 ratio is increased after Vps35 knockdown. ELISA analysis was carried out on media from control and Vps35 knockdown 293 695 cultures to detect levels of secreted Aβ40 and Aβ42. Aβ levels were detected and normalized against cellular protein levels. The ratio of Aβ42 to Aβ40 was also determined for each cell line. Relative values from a representative experiment are shown. The difference in Aβ40 levels between control and Vps35 knockdown cells is significant (P
    Figure Legend Snippet: Aβ40 secretion is decreased and the Aβ42:Aβ40 ratio is increased after Vps35 knockdown. ELISA analysis was carried out on media from control and Vps35 knockdown 293 695 cultures to detect levels of secreted Aβ40 and Aβ42. Aβ levels were detected and normalized against cellular protein levels. The ratio of Aβ42 to Aβ40 was also determined for each cell line. Relative values from a representative experiment are shown. The difference in Aβ40 levels between control and Vps35 knockdown cells is significant (P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    18) Product Images from "Dissociable cognitive impairments in two strains of transgenic Alzheimer’s disease mice revealed by a battery of object-based tests"

    Article Title: Dissociable cognitive impairments in two strains of transgenic Alzheimer’s disease mice revealed by a battery of object-based tests

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37312-0

    Aβ42 deposition in 13 month old 5xFAD and 3xTG mice. ( a ) There is extensive Aβ42 accumulation in the cortex and HPC of 5xFAD mice (n = 4 per group; main effects of genotype (F 1,24 = 632.278, p
    Figure Legend Snippet: Aβ42 deposition in 13 month old 5xFAD and 3xTG mice. ( a ) There is extensive Aβ42 accumulation in the cortex and HPC of 5xFAD mice (n = 4 per group; main effects of genotype (F 1,24 = 632.278, p

    Techniques Used: Mouse Assay

    19) Product Images from "Phenolic Compounds Prevent Alzheimer's Pathology through Different Effects on the Amyloid-? Aggregation Pathway"

    Article Title: Phenolic Compounds Prevent Alzheimer's Pathology through Different Effects on the Amyloid-? Aggregation Pathway

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2009.090417

    Assessment of Aβ40 ( A ), Aβ42 ( B ), Aβ40 + Aβ42 ( C ), and aggregated Aβ ( D ) concentrations in the TBS-soluble fraction of mice brain tissue. There was no significant difference between the control and treatment
    Figure Legend Snippet: Assessment of Aβ40 ( A ), Aβ42 ( B ), Aβ40 + Aβ42 ( C ), and aggregated Aβ ( D ) concentrations in the TBS-soluble fraction of mice brain tissue. There was no significant difference between the control and treatment

    Techniques Used: Mouse Assay

    Assessment of Aβ40 ( A ), Aβ42 ( B ), and Aβ40 + Aβ42 ( C ) concentrations in the TBS-insoluble fraction of mice brain tissue. Compared with the control, Aβ40 was significantly reduced in the Cur ( P
    Figure Legend Snippet: Assessment of Aβ40 ( A ), Aβ42 ( B ), and Aβ40 + Aβ42 ( C ) concentrations in the TBS-insoluble fraction of mice brain tissue. Compared with the control, Aβ40 was significantly reduced in the Cur ( P

    Techniques Used: Mouse Assay

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: A Becn1 mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in Alzheimer's disease
    Article Snippet: To measure Aβ levels in conditioned media of APP-HEK293 cells, media of 72-h cell culture was collected, mixed with 4X sample buffer (50 mM Tris-HCl pH6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue), and boiled at 95°C for 10 min. One μl of each sample was spotted on nitrocellulose membrane for dot blot analysis. .. ELISA GuHCl extracted brain samples prepared in the same way as dot blot assays were diluted 1:1000, and ELISA analyses of Aβ42 were performed according to manufacturer’s instructions (Thermo Fisher Scientific, KHB3441). .. Immunofluorescence microscopy Paraformaldehyde-fixed brain tissues were sectioned at 30 μm thickness.

    Article Title: Suppression of glymphatic fluid transport in a mouse model of Alzheimer’s disease
    Article Snippet: We used 125 I-Aβ40 without added aprotinin, a potential inhibitor of LRP1-mediated transport. .. ELISA kits for Aβ40 (KHB 3481) and Aβ42 (KHB 3441) were obtained from Invitrogen (Camarilla, CA, USA) and Aβ oligomer ELISA kit (BEK-2215-1P) from Biosensis (Termecula, CA, USA). .. Mice were anesthetized as indicated above, fixed to a stereotactic frame, cisterna magna exposed and cannulated with a 30G needle ( ; ).

    Article Title: Rapid and sustained cognitive recovery in APP/PS1 transgenic mice by co-administration of EPPS and donepezil
    Article Snippet: Hippocampal and cortical regions of the brain were dissected separately and homogenized in ice-cold lysis buffer, RIPA, containing 1x protease inhibitors cocktail. .. The supernatant (soluble fraction) of brain lysates was analyzed with Aβ42 ELISA kit purchased from Invitrogen (KHB3442). .. To obtain Aβ insoluble fraction in brain lysates, a guanidine buffer (5 mM guanidine-HCl, 50 mM Tris-HCl, pH 8.0) containing 1x protease inhibitors cocktail was added to the pellet of brains lysates and incubated at room temperature for 3 hr with shaking.

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    Article Title: Suppression of glymphatic fluid transport in a mouse model of Alzheimer’s disease
    Article Snippet: The supernatant was used to determine levels of soluble Aβ oligomers. .. Levels (pmol/g brain tissue) of human Aβ40, Aβ42 and soluble Aβ oligomers were determined using ELISA kits [Aβ40 (KHB 3481) and Aβ42 (KHB 3441); Invitrogen (Camarilla, CA, USA] and following the manufacturers’ instructions. .. Since soluble Aβ oligomers vary in size and number of oligomers the unit is arbitrary and is indicated as equivalent to Aβ.

    Article Title: Blocking beta 2-adrenergic receptor inhibits dendrite ramification in a mouse model of Alzheimer's disease
    Article Snippet: The average number of dendritic branches of pyramidal neurons in the hippocampus was analyzed by Golgi staining. .. Enzyme linked immunosorbent assay Amyloid beta 40 (Aβ40 ) and amyloid beta 42 (Aβ42 ) enzyme linked immunosorbent assay plates were purchased from Invitrogen (Waltham, MA, USA) and experiments were conducted according to the manufacturer's instructions. .. Western blot assay Proteins (20 μg) from each sample of hippocampus were loaded on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel.

    Article Title: Protective effects of 7,8-dihydroxyflavone on neuropathological and neurochemical changes in a mouse model of Alzheimer’s disease
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    Dot Blot:

    Article Title: A Becn1 mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in Alzheimer's disease
    Article Snippet: To measure Aβ levels in conditioned media of APP-HEK293 cells, media of 72-h cell culture was collected, mixed with 4X sample buffer (50 mM Tris-HCl pH6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue), and boiled at 95°C for 10 min. One μl of each sample was spotted on nitrocellulose membrane for dot blot analysis. .. ELISA GuHCl extracted brain samples prepared in the same way as dot blot assays were diluted 1:1000, and ELISA analyses of Aβ42 were performed according to manufacturer’s instructions (Thermo Fisher Scientific, KHB3441). .. Immunofluorescence microscopy Paraformaldehyde-fixed brain tissues were sectioned at 30 μm thickness.

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    Thermo Fisher aβ42
    Gray matter of UCSD immunized cases separated by HPLC (C8 reverse-phase) . The figure shows the HPLC chromatogram fractions that were investigated by Western blotting developed with anti-Aβ40 and <t>anti-Aβ42</t> and CT9APP antibodies. The diagonal-hyphenated line represents the acetonitrile gradient. Lanes 10 contain the standards Aβ40 or Aβ42 synthetic peptides. A) and B), correspond to the neuropathologically confirmed AD cases # 19 and # 20, respectively. C) corresponds to the neuropathologically confirmed hippocampal sclerosis. m, Aβ monomer; d, Aβ dimer; t, Aβ trimer.
    Aβ42, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti aβ42
    Rapamycin reduces Aβ levels in 7AP2 cells. A , representative Western blots of proteins extracted from 7PA2 cells treated with different concentrations of rapamycin for 24 h. B , densitometric analysis of the blots (normalized to β-actin) shows that rapamycin had no effect on total p70S6K levels but completely blocked phosphorylation of p70S6K at Thr 389 ( n = 9; horizontal line ). C , rapamycin rescued the increased mTOR enzymatic activity in 7PA2 cells ( n = 9). The horizontal line shows the levels of mTOR activity in control. D , sandwich ELISA measurements of proteins extracted from 7PA2 cells treated with rapamycin show that at all concentrations used, rapamycin significantly decreased the <t>Aβ42</t> levels ( n = 9). All the experiments shown here were done in triplicates in three independent experiments; thus, we analyzed a total of 9 samples for each cell line, for each specific condition. Data are presented as mean ± S.E. Protein levels are expressed as arbitrary units. * indicates p
    Anti Aβ42, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gray matter of UCSD immunized cases separated by HPLC (C8 reverse-phase) . The figure shows the HPLC chromatogram fractions that were investigated by Western blotting developed with anti-Aβ40 and anti-Aβ42 and CT9APP antibodies. The diagonal-hyphenated line represents the acetonitrile gradient. Lanes 10 contain the standards Aβ40 or Aβ42 synthetic peptides. A) and B), correspond to the neuropathologically confirmed AD cases # 19 and # 20, respectively. C) corresponds to the neuropathologically confirmed hippocampal sclerosis. m, Aβ monomer; d, Aβ dimer; t, Aβ trimer.

    Journal: Molecular Neurodegeneration

    Article Title: The biochemical aftermath of anti-amyloid immunotherapy

    doi: 10.1186/1750-1326-5-39

    Figure Lengend Snippet: Gray matter of UCSD immunized cases separated by HPLC (C8 reverse-phase) . The figure shows the HPLC chromatogram fractions that were investigated by Western blotting developed with anti-Aβ40 and anti-Aβ42 and CT9APP antibodies. The diagonal-hyphenated line represents the acetonitrile gradient. Lanes 10 contain the standards Aβ40 or Aβ42 synthetic peptides. A) and B), correspond to the neuropathologically confirmed AD cases # 19 and # 20, respectively. C) corresponds to the neuropathologically confirmed hippocampal sclerosis. m, Aβ monomer; d, Aβ dimer; t, Aβ trimer.

    Article Snippet: The ELISA kits to quantify Aβ40 and Aβ42 were obtained from Invitrogen (Carlsbad, CA) and Innogenetics (Gent, Belgium), respectively, and performed following the manufacturer's instructions.

    Techniques: High Performance Liquid Chromatography, Western Blot

    Chronic ICI administration exacerbates Aβ pathology (A–D) Representative photomicrographs of 3xTg-AD mice treated with ICI or NaCl. Sections were immunostained with an Aβ42-specific antibody from Millipore. Panels C and D are higher magnification views of panels A and B, respectively. (E) The graph shows average number of plaques in each brain section from treated and untreated 3xTg-AD mice. (F) ELISA measurements from brain lysates revealed no difference in both soluble and insoluble Aβ40 levels. (G) In contrast, insoluble Aβ42 levels were significantly increased in the brains of ICI-treated 3xTg-AD mice (p

    Journal: Neurobiology of aging

    Article Title: Administration of a selective β2 adrenergic receptor antagonist exacerbates neuropathology and cognitive deficits in a mouse model of Alzheimer's disease

    doi: 10.1016/j.neurobiolaging.2014.06.011

    Figure Lengend Snippet: Chronic ICI administration exacerbates Aβ pathology (A–D) Representative photomicrographs of 3xTg-AD mice treated with ICI or NaCl. Sections were immunostained with an Aβ42-specific antibody from Millipore. Panels C and D are higher magnification views of panels A and B, respectively. (E) The graph shows average number of plaques in each brain section from treated and untreated 3xTg-AD mice. (F) ELISA measurements from brain lysates revealed no difference in both soluble and insoluble Aβ40 levels. (G) In contrast, insoluble Aβ42 levels were significantly increased in the brains of ICI-treated 3xTg-AD mice (p

    Article Snippet: Aβ40 and Aβ42 ELISA plates were purchased from Invitrogen and experiments were conducted using the manufacturer’s instructions.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    The pharmacological inhibitor of DAPK1 reduces the secretion of Aβ40 and Aβ42. ( A , B ) Human H4 (A) and SH-SY5Y (B) cells were treated with the indicated concentrations of DAPK1 pharmacological inhibitor for 24 h. Levels of human Aβ40 in cell culture supernatants after 24 h incubation were determined by a solid phase sandwich ELISA assay as in (B) and ( C ). Each data point represents mean ± standard error of three independent experiments (* P

    Journal: Human Molecular Genetics

    Article Title: Inhibition of death-associated protein kinase 1 attenuates the phosphorylation and amyloidogenic processing of amyloid precursor protein

    doi: 10.1093/hmg/ddw114

    Figure Lengend Snippet: The pharmacological inhibitor of DAPK1 reduces the secretion of Aβ40 and Aβ42. ( A , B ) Human H4 (A) and SH-SY5Y (B) cells were treated with the indicated concentrations of DAPK1 pharmacological inhibitor for 24 h. Levels of human Aβ40 in cell culture supernatants after 24 h incubation were determined by a solid phase sandwich ELISA assay as in (B) and ( C ). Each data point represents mean ± standard error of three independent experiments (* P

    Article Snippet: Aβ secretion into the medium was determined using mouse Aβ40, mouse Aβ42, human Aβ40 and human Aβ42 colorimetric ELISA Kits (all from Invitrogen), following the manufacturer’s instructions.

    Techniques: Cell Culture, Incubation, Sandwich ELISA

    DAPK1 inhibition reduces the secretion of human Aβ40 and Aβ42 from both SH-SY5Y APPwt and SH-SY5Y APPswe cells. ( A ) Baseline expression of APP in parent SH-SY5Y cells and SH-SY5Y cells stably expressing empty vector (pcDNA3), APP WT (pcDNA3-hAPP 695 ) or APP Swedish mutant (pcDNA3-hAPP 695swe ). The cell lysates were subjected to western blot analysis with anti-APP or anti-tubulin antibody. The blots are representative of three independent experiments. ( B , C ) SH-SY5Y APPwt and SH-SY5Y APPswe cells were transfected with three different human DAPK1-sepcific siRNAs or scrambled control siRNA for 60 h. The cell lysates were subjected to western blot analysis with anti-DAPK1 or anti-tubulin antibody and the supernatants were subjected to human Aβ40 and Aβ42 ELISA analyses. The blots are representative of three independent experiments and ELISA data shown represent the mean ± standard error of three independent experiments (* P

    Journal: Human Molecular Genetics

    Article Title: Inhibition of death-associated protein kinase 1 attenuates the phosphorylation and amyloidogenic processing of amyloid precursor protein

    doi: 10.1093/hmg/ddw114

    Figure Lengend Snippet: DAPK1 inhibition reduces the secretion of human Aβ40 and Aβ42 from both SH-SY5Y APPwt and SH-SY5Y APPswe cells. ( A ) Baseline expression of APP in parent SH-SY5Y cells and SH-SY5Y cells stably expressing empty vector (pcDNA3), APP WT (pcDNA3-hAPP 695 ) or APP Swedish mutant (pcDNA3-hAPP 695swe ). The cell lysates were subjected to western blot analysis with anti-APP or anti-tubulin antibody. The blots are representative of three independent experiments. ( B , C ) SH-SY5Y APPwt and SH-SY5Y APPswe cells were transfected with three different human DAPK1-sepcific siRNAs or scrambled control siRNA for 60 h. The cell lysates were subjected to western blot analysis with anti-DAPK1 or anti-tubulin antibody and the supernatants were subjected to human Aβ40 and Aβ42 ELISA analyses. The blots are representative of three independent experiments and ELISA data shown represent the mean ± standard error of three independent experiments (* P

    Article Snippet: Aβ secretion into the medium was determined using mouse Aβ40, mouse Aβ42, human Aβ40 and human Aβ42 colorimetric ELISA Kits (all from Invitrogen), following the manufacturer’s instructions.

    Techniques: Inhibition, Expressing, Stable Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay

    DAPK1 KO reduces the secretion of Aβ40 and Aβ42 in mouse models. ( A ) Whole brain lysates from WT and DAPK1 KO mice harvested at 6 month of age were analyzed for the levels of phosphorylated (Thr668 and Tyr682) and total APP protein as well as DAPK1. Anti-actin antibody was used as a loading control. The blots are representative of three independent experiments. Analyzed mice number: WT ( n = 6, 3 male (M) and 3 female (F)), DAPK1 KO (3M, 3F). ( B , C ) Primary cortical neurons (B) at DIV 8-10 of WT and DAPK1 KO mice and the brain hippocampal tissue slices (C) prepared from WT and DAPK1 KO mice at 6 month of age were cultured for 36 h. Levels of mouse Aβ40 in cell culture supernatants after 36 h incubation were determined by a solid phase sandwich ELISA assay. Each data point represents mean ± standard error of three independent experiments (* P

    Journal: Human Molecular Genetics

    Article Title: Inhibition of death-associated protein kinase 1 attenuates the phosphorylation and amyloidogenic processing of amyloid precursor protein

    doi: 10.1093/hmg/ddw114

    Figure Lengend Snippet: DAPK1 KO reduces the secretion of Aβ40 and Aβ42 in mouse models. ( A ) Whole brain lysates from WT and DAPK1 KO mice harvested at 6 month of age were analyzed for the levels of phosphorylated (Thr668 and Tyr682) and total APP protein as well as DAPK1. Anti-actin antibody was used as a loading control. The blots are representative of three independent experiments. Analyzed mice number: WT ( n = 6, 3 male (M) and 3 female (F)), DAPK1 KO (3M, 3F). ( B , C ) Primary cortical neurons (B) at DIV 8-10 of WT and DAPK1 KO mice and the brain hippocampal tissue slices (C) prepared from WT and DAPK1 KO mice at 6 month of age were cultured for 36 h. Levels of mouse Aβ40 in cell culture supernatants after 36 h incubation were determined by a solid phase sandwich ELISA assay. Each data point represents mean ± standard error of three independent experiments (* P

    Article Snippet: Aβ secretion into the medium was determined using mouse Aβ40, mouse Aβ42, human Aβ40 and human Aβ42 colorimetric ELISA Kits (all from Invitrogen), following the manufacturer’s instructions.

    Techniques: Mouse Assay, Cell Culture, Incubation, Sandwich ELISA

    Rapamycin reduces Aβ levels in 7AP2 cells. A , representative Western blots of proteins extracted from 7PA2 cells treated with different concentrations of rapamycin for 24 h. B , densitometric analysis of the blots (normalized to β-actin) shows that rapamycin had no effect on total p70S6K levels but completely blocked phosphorylation of p70S6K at Thr 389 ( n = 9; horizontal line ). C , rapamycin rescued the increased mTOR enzymatic activity in 7PA2 cells ( n = 9). The horizontal line shows the levels of mTOR activity in control. D , sandwich ELISA measurements of proteins extracted from 7PA2 cells treated with rapamycin show that at all concentrations used, rapamycin significantly decreased the Aβ42 levels ( n = 9). All the experiments shown here were done in triplicates in three independent experiments; thus, we analyzed a total of 9 samples for each cell line, for each specific condition. Data are presented as mean ± S.E. Protein levels are expressed as arbitrary units. * indicates p

    Journal: The Journal of Biological Chemistry

    Article Title: Molecular Interplay between Mammalian Target of Rapamycin (mTOR), Amyloid-?, and Tau

    doi: 10.1074/jbc.M110.100420

    Figure Lengend Snippet: Rapamycin reduces Aβ levels in 7AP2 cells. A , representative Western blots of proteins extracted from 7PA2 cells treated with different concentrations of rapamycin for 24 h. B , densitometric analysis of the blots (normalized to β-actin) shows that rapamycin had no effect on total p70S6K levels but completely blocked phosphorylation of p70S6K at Thr 389 ( n = 9; horizontal line ). C , rapamycin rescued the increased mTOR enzymatic activity in 7PA2 cells ( n = 9). The horizontal line shows the levels of mTOR activity in control. D , sandwich ELISA measurements of proteins extracted from 7PA2 cells treated with rapamycin show that at all concentrations used, rapamycin significantly decreased the Aβ42 levels ( n = 9). All the experiments shown here were done in triplicates in three independent experiments; thus, we analyzed a total of 9 samples for each cell line, for each specific condition. Data are presented as mean ± S.E. Protein levels are expressed as arbitrary units. * indicates p

    Article Snippet: The following antibodies were from the indicated sources: rabbit polyclonal anti-LC3 (Novus Biologicals, Littlelton, CO); anti-Actin (Sigma); anti-mTOR, phospho-mTOR, Phospho-p70S6K (Thr389 ), p70S6K, anti-ATG5, and anti-ATG7 (Cell Signaling, Boston, MA); anti-Tau HT7 and anti-AT270 (Pierce); anti-Aβ42 (Invitrogen); 6E10 (Signet, Dedham, MA); anti-Tau MC1 was a gift from Dr. Peter Davies; and anti-Lamp2A (Abcam, Cambridge, MA). mTOR activity was measured using the K-LISATM mTOR Activity Kit (EMD Chemicals, Gibbstown, NJ) following the manufacturer's protocol.

    Techniques: Western Blot, Activity Assay, Sandwich ELISA