anti adam10 polyclonal antibody  (R&D Systems)

Journal: Alzheimer's Research & Therapy

Article Title: Selective reduction of ADAM10 in brain and cerebrospinal fluid of Alzheimer’s disease patients

doi: 10.1186/s13195-026-02007-6

Figure Lengend Snippet: Protein levels of ADAM10 species and mRNA in AD brain samples. Non-dementia controls (NDC, n = 13) and AD extracts ( n = 16) from prefrontal cortex were resolved by SDS-PAGE/electrophoresis prior to western blot assay. Each individual ADAM10 immunoreactive band was quantified, and levels normalized using GAPDH. A Representative western blot of ADAM10 using the rabbit anti- C-terminal region monoclonal antibody (ab124695, Abcam) and GAPDH with monoclonal antibody (60004-1-Ig, Proteintech) ( B ) Densitometric quantification of ADAM10 immunoreactive bands assigned to immature (iADAM10) and C mature (mADAM10) species normalized with respect to GAPDH. D Ratio mADAM10/iADAM10 that represents the amount of mature species vs. immature. E Relative mRNA levels of ADAM10 in NDC vs. AD patients’ samples were analyzed by qRT-PCR. Transcript levels were calculated by the comparative 2 − ΔCt method with respect to GAPDH. The graphs represent mean ± SEM. Significant P < 0.05 values assayed by t-test are indicated

Article Snippet: Representative blots of CSF samples of non-AD control individuals with large age amplitude (the age of the subjects are shown on top) probed with ( A ) anti-ADAM10 polyclonal antibody to ectodomain region (OAGA02442, Aviva) and ( B ) ADAM17 antibody towards the ectodomain region (AF9301, R&D Systems).

Techniques: SDS Page, Electrophoresis, Western Blot, Quantitative RT-PCR

Journal: Alzheimer's Research & Therapy

Article Title: Selective reduction of ADAM10 in brain and cerebrospinal fluid of Alzheimer’s disease patients

doi: 10.1186/s13195-026-02007-6

Figure Lengend Snippet: Aβ affects ADAM10 levels, but not ADAM17, in SH-SY5Y-differentiated neurons. SH-SY5Y cell cultures were differentiated to neurons with 10 µM retinoic acid treatment and then treated with 3 µM of Aβ42 for 48 h. A Representative western blot of control ( C ) and Aβ-treated (Aβ42) cell samples that was resolved with the anti C-terminal ADAM10 antibody (ab124695, Abcam). B Densitometric quantification of immunoreactive bands of iADAM10 and C mADAM10 with respect to GAPDH. Values represent the percentage with respect to control. D Values of the ratio between mature vs. immature species of ADAM10 (mADAM10/iADAM10). E Representative western blot of ADAM17 species resolved with anti-ectodomain region ADAM17 antibody (AF9301, R&D Systems) and the respective quantifications of F iADAM17 and G mADAM17 immunoreactive bands normalized to the ubiquitous protein GAPDH. H Result of the ratio of mADAM17/iADAM17. The graphs represent mean ± SEM of n = 12 samples of 3 independent experiments. Significant P < 0.05 values assayed by t-test are indicated

Article Snippet: Representative blots of CSF samples of non-AD control individuals with large age amplitude (the age of the subjects are shown on top) probed with ( A ) anti-ADAM10 polyclonal antibody to ectodomain region (OAGA02442, Aviva) and ( B ) ADAM17 antibody towards the ectodomain region (AF9301, R&D Systems).

Techniques: Western Blot, Control

Journal: Alzheimer's Research & Therapy

Article Title: Selective reduction of ADAM10 in brain and cerebrospinal fluid of Alzheimer’s disease patients

doi: 10.1186/s13195-026-02007-6

Figure Lengend Snippet: ADAM10 and ADAM17 CSF levels are not altered with aging. Representative blots of CSF samples of non-AD control individuals with large age amplitude (the age of the subjects are shown on top) probed with ( A ) anti-ADAM10 polyclonal antibody to ectodomain region (OAGA02442, Aviva) and ( B ) ADAM17 antibody towards the ectodomain region (AF9301, R&D Systems). Correlations between the age and the levels of ADAM10 and ADAM17 species ( C ) iADAM10, ( D ) mADAM10, ( E ) sADAM10, ( F ) iADAM17, ( G ) mADAM17 and ( H ) sADAM17 were assayed. All graphs include their Spearman coefficient (r) and the P value. None of the ADAM10 species nor ADAM17 correlate with age. * Refers to unspecific bands

Article Snippet: Representative blots of CSF samples of non-AD control individuals with large age amplitude (the age of the subjects are shown on top) probed with ( A ) anti-ADAM10 polyclonal antibody to ectodomain region (OAGA02442, Aviva) and ( B ) ADAM17 antibody towards the ectodomain region (AF9301, R&D Systems).

Techniques: Control

Journal: Alzheimer's Research & Therapy

Article Title: Selective reduction of ADAM10 in brain and cerebrospinal fluid of Alzheimer’s disease patients

doi: 10.1186/s13195-026-02007-6

Figure Lengend Snippet: Levels of ADAM10 and ADAM17 species in AD CSF samples. Analysis of CSF ADAM10 and ADAM17 in non-AD controls (NADC) and in AD patients. A Representative blot of CSF probed against anti-ADAM10 ectodomain antibody. Quantification of immunoreactive band values obtained from B iADAM10, C mADAM10 and D sADAM10. E Values of the ratio between mature vs immature species of ADAM10 (mADAM10/iADAM10). F Representative blot of CSF probed against ADAM17 and the quantifications of the immunoreactivity of the bands for G iADAM17, H mADAM17 and I sADAM17. J Result of the ratio mADAM10/iADAM10. The graphs represent mean ± SEM. Significant P < 0.05 values assayed by t-test are indicated. * Refers to unspecific bands

Article Snippet: Representative blots of CSF samples of non-AD control individuals with large age amplitude (the age of the subjects are shown on top) probed with ( A ) anti-ADAM10 polyclonal antibody to ectodomain region (OAGA02442, Aviva) and ( B ) ADAM17 antibody towards the ectodomain region (AF9301, R&D Systems).

Techniques:

Journal: Frontiers in Immunology

Article Title: A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers

doi: 10.3389/fimmu.2024.1434027

Figure Lengend Snippet: AD inhibits ADAM10 expression in various cancer cells. (A) AD inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the H1975 lung cancer cells. (B) AD inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the A549 lung cancer cells. (C) AD inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the BT549 breast cancer cells. (D) Quantitative results for ADAM10 protein levels. (E) Quantitative results for ADAM10 mRNA levels. AD, adenosine; ADAM10, a disintegrin and metalloproteinase domain 10. *P <0.05; **P < 0.005; ****P < 0.0001.

Article Snippet: Anti-ADAM10 polyclonal antibody was used at a dilution of 1:4,000. β-actin (Cat No6609–1-Ig;Proteintech, Wuhan, China) served as an internal control.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers

doi: 10.3389/fimmu.2024.1434027

Figure Lengend Snippet: Cordycepin (CD) inhibits ADAM10 expressions in various cancer cells. (A) CD inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the H1975 lung cancer cell line. (B) CD inhibited expression of ADAM10 in protein showing in upper panel and mRNA showing in lower panel in the A549 lung cancer cell line. (C) CD inhibited expression of ADAM10 in protein showing in upper panel and mRNA showing in lower panel in the BT549 breast cancer cell line. (D) Quantitative results for ADAM10 protein levels. (E) Quantitative results for ADAM10 mRNA levels. CD, cordycepin; ADAM10, a disintegrin and metalloproteinase domain 10. *P <0.05; ****P < 0.0001.

Article Snippet: Anti-ADAM10 polyclonal antibody was used at a dilution of 1:4,000. β-actin (Cat No6609–1-Ig;Proteintech, Wuhan, China) served as an internal control.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers

doi: 10.3389/fimmu.2024.1434027

Figure Lengend Snippet: m 6 2 A inhibits ADAM10 expressions in various cancer cell lines. (A) m 6 2 A inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the H1975 lung cancer cell line. (B) m 6 2 A inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the A549 lung cancer cell line. (C) m 6 2 A inhibited expression of ADAM10 in both protein showing in upper panel and mRNA showing in lower panel in the BT549 breast cancer cell line. (D) Quantitative results for ADAM10 protein levels. (E) Quantitative results for ADAM10 mRNA levels. m 6 2 A, N6, N6-dimethyladenosine; ADAM10, a disintegrin and metalloproteinase domain 10. ****P < 0.0001.

Article Snippet: Anti-ADAM10 polyclonal antibody was used at a dilution of 1:4,000. β-actin (Cat No6609–1-Ig;Proteintech, Wuhan, China) served as an internal control.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers

doi: 10.3389/fimmu.2024.1434027

Figure Lengend Snippet: ADAM10 stability by AD and CD. (A) ADAM10 stability by AD treatments. Right panel, quantitative results. (B) ADAM10 stability by CD treatments. Right panel, quantitative results. CHX (20µg/ml) was used to treat A549 lung cancer cell lines or BT549 breast cancer cell lines with or without indicated AD or CD treatments, and western blot was performed. β-actin was used as an internal control. CHX, cycloheximide.

Article Snippet: Anti-ADAM10 polyclonal antibody was used at a dilution of 1:4,000. β-actin (Cat No6609–1-Ig;Proteintech, Wuhan, China) served as an internal control.

Techniques: Western Blot, Control

Journal: Frontiers in Immunology

Article Title: A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers

doi: 10.3389/fimmu.2024.1434027

Figure Lengend Snippet: ADAM10 (6bdz) molecular docking binding sites and energy.

Article Snippet: Anti-ADAM10 polyclonal antibody was used at a dilution of 1:4,000. β-actin (Cat No6609–1-Ig;Proteintech, Wuhan, China) served as an internal control.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers

doi: 10.3389/fimmu.2024.1434027

Figure Lengend Snippet: Molecular docking for ADAM10 (6bdz) and AD, CD, and m 6 2 A binding sites. (A) Molecular docking of AD with human ADAM10 protein (6bdz) and active site residues and (B) force of attractions involved in docking; (C) Molecular docking of CD with human ADAM10 protein (6bdz) and active site residues and (D) force of attractions involved in docking; (E) Molecular docking of AD with human ADAM10 protein (6bdz) and active site residues and (F) force of attractions involved in docking.

Article Snippet: Anti-ADAM10 polyclonal antibody was used at a dilution of 1:4,000. β-actin (Cat No6609–1-Ig;Proteintech, Wuhan, China) served as an internal control.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers

doi: 10.3389/fimmu.2024.1434027

Figure Lengend Snippet: Bioinformatic analysis for the immunoregulatory actions of ADAM10 in various cancers. (A) Correlations between ADAM10 and 150 genes of Immunoregulation. There are 41 chemokines, 18 receptors, 21 MHCs, 24 immunoinhibitors, and 46 immunostimulators. (B) Correlations between ADAM10 and 60 genes of immune checkpoint pathways. (C) AD inhibits LAG3 expression in H1975 cells. Up panel indicates semi-quantitative RT-PCR, bottom panel indicates the quantitative results from up panel. (D) CD inhibits LAG3 expression in BT549 cells. Up panel indicates semi-quantitative RT-PCR, bottom panel indicates the quantitative results from up panel. *P <0.05. ADAM10, a disintegrin and metalloproteinase domain 10. For immunomodulatory genetic analysis, the pan-cancer data-set The Cancer Genome Atlas (TCGA) TARGET Genotype-Tissue Expression (GTEx) (PANCAN) was downloaded from University of California Santa Cruz (UCSC; https://xenabrowser.net/ ).

Article Snippet: Anti-ADAM10 polyclonal antibody was used at a dilution of 1:4,000. β-actin (Cat No6609–1-Ig;Proteintech, Wuhan, China) served as an internal control.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers

doi: 10.3389/fimmu.2024.1434027

Figure Lengend Snippet: Pearson’s correlations of the correlation between ADAM10 expression and tumor-related immune cells in various cancers calculated using different methods. (A) Correlation between ADAM10 and six tumor-associated immune cells calculated using Tumor Immune Estimation Resource. (B) Correlations between ADAM10 and six tumor-related immune cells calculated using deconvo_ips. (C) Correlations between ADAM10 and 22 tumor-associated immune cells calculated using deconvo CIBERSOR. *P <0.05; **P < 0.005; ***P <0.001; ****P < 0.0001. ADAM10, a disintegrin and metalloproteinase domain 10.

Article Snippet: Anti-ADAM10 polyclonal antibody was used at a dilution of 1:4,000. β-actin (Cat No6609–1-Ig;Proteintech, Wuhan, China) served as an internal control.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers

doi: 10.3389/fimmu.2024.1434027

Figure Lengend Snippet: Association between ADAM10 expression and immune infiltration score in multiple types of cancer. For immune infiltration analysis, the pan-cancer data-set TCGA TARGET Genotype-Tissue Expression (GTEx) was also downloaded from UCSC. ADAM10 expression and immune infiltration score in multiple types of cancer are presented in the indicated different panels.

Article Snippet: Anti-ADAM10 polyclonal antibody was used at a dilution of 1:4,000. β-actin (Cat No6609–1-Ig;Proteintech, Wuhan, China) served as an internal control.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers

doi: 10.3389/fimmu.2024.1434027

Figure Lengend Snippet: Immuno-infiltration analysis of ADAM10 gene in pan-cancer.

Article Snippet: Anti-ADAM10 polyclonal antibody was used at a dilution of 1:4,000. β-actin (Cat No6609–1-Ig;Proteintech, Wuhan, China) served as an internal control.

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: The mature active form of ADAM10 is increased in the HD mouse hippocampus and causes N-CAD proteolysis. A Representative Western blot for the mature active form of ADAM10 (m-ADAM10) in synaptosomal fractions obtained from the hippocampus of R6/2 transgenic mice and zQ175 heterozygous knock-in mice. β-III Tubulin, loading control. B Quantification of data shown in A. WT and R6/2 mice at 10–12 weeks of age: n=12–13 mice/genotype. WT and zQ175 mice at 54 weeks of age: n=9 mice/genotype. Data are represented as mean ± SEM. ****P < 0.0001, unpaired t test. C Representative Western blot of N-CAD-CTF in the hippocampus from WT and HD mice (R6/2 and zQ175). α-Tubulin, loading control. D Quantification of results shown in C. The N-CAD-CTF signal intensity has been divided for the FL N-CAD content, which has been determined by dividing FL N-CAD intensity over the α-Tubulin intensity. WT and R6/2 mice at 10–12 weeks of age: n=8–9 mice/genotype; WT and zQ175 mice at 54 weeks: n=3 mice/genotype. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, unpaired t test

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques: Western Blot, Transgenic Assay, Knock-In, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: ADAM10 heterozygous deletion in the forebrain rescues dendritic spine loss in the CA1 region of the hippocampus in R6/2 mice. A Representative examples of secondary apical dendritic segments of CA1 pyramidal neurons from 13-week-old WT, R6/2, R6/2-A10cKO and A10cKO mice. Scale bars: 10 µm, 80 × Objective. S, stubby spines; M, mushroom spines; T, thin spines. B Total dendritic spine density. C Stubby spine density. D Mushroom spine density. E Thin spine density. In B-E n = 3 mice/genotype were analyzed for a total of n = 30 neurons/genotype. Each dot in the graphs represents the mean ± SEM of the spine density in 10 µm dendrite for each neuron analyzed. *P < 0.05, ****P < 0.0001, One-way ANOVA with Tukey’s post hoc test

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: ADAM10 heterozygous deletion in the forebrain rescues ultrastructural defects of the HD hippocampal synapse. A Diagram showing SVs classification based on distance from the presynaptic membrane (docked: 0–50 nm, reserve: 50–300 nm, resting: > 300 nm) with corresponding tenuous background colors added as a guide for the eye in TEM images reported in panel ( B ). B Representative TEM images of excitatory synapses in pyramidal neurons of the CA1 region of the hippocampus of WT, R6/2, R6/2-A10cKO and A10cKO mice at 13 weeks of age. Scale bars: 100 nm. PSD, post-synaptic density. C Density of total SVs. D Density of docked SVs. E Density of reserve SVs. F Density of resting SVs. In C-F, n = 3 mice/genotype and n = 60 excitatory synapses/genotype were analyzed. Each dot in the graphs represents the n° SVs/µm 2 for each excitatory synapse analyzed. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Kruskal–Wallis with Dunn’s multiple comparisons test

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques: Membrane

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: ADAM10 heterozygous deletion in the forebrain enhances BDNF synthesis and promotes ERK phosphorylation in the R6/2 hippocampus. A Scheme of the mouse BDNF gene and BDNF mRNA isoforms. B Total BDNF mRNA level and level of BDNF mRNA isoforms in the hippocampus of WT, R6/2 and R6/2-A10cKO mice at 13 weeks of age. WT: n = 4–7; R6/2: n = 5–7; R6/2-A10cKO: n = 7–8. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA with Bonferroni’s post hoc test. For BDNF mRNA isoform II the forward and reverse primers (see Methods) led to simultaneous amplification of the transcript variant IIA, IIB, and IIC. C ELISA for BDNF in the hippocampus of WT, R6/2 and R6/2-A10cKO mice at 13 weeks of age. WT: n = 4; R6/2: n = 6; R6/2-A10cKO: n = 9. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, One-way ANOVA with Bonferroni’s post hoc test. D Representative Western blot for total and phosphorylated ERK1/2 in the hippocampus of WT, R6/2 and R6/2-A10cKO mice at 13 weeks of age. β-III Tubulin, loading control. E , F Quantification of data in D. WT: n = 5; R6/2: n = 8; R6/2-A10cKO: n = 11. Data are represented as mean ± SEM. **P < 0.01, ***P < 0.001, One-way ANOVA with Bonferroni’s post hoc test

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques: Amplification, Variant Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: TrkB mediates the neuroprotective effect determined by ADAM10 inhibition on long-lasting spine loss in HD hippocampal neurons. A Hippocampal neurons from WT and R6/2 mice were transfected at DIV5 with pcDNA3.1-mGreenLantern plasmid. The ADAM10 inhibitor GI254023X (GI, 1 µM) was administered from DIV6 until DIV14. The TrkB antagonist ANA12 (10 µM) was administered at DIV12 and cells were fixed at DIV14 for spine analyses and excitatory synapses quantification. B Immunofluorescence images of dendritic spines in hippocampal cultures: WT, WT + ANA12, R6/2, R6/2 + ANA12, R6/2 + GI; R6/2 + GI + ANA12. Scale bars: 10 µm. M, mushroom spines; T, thin spines; S, stubby spines. C-F Density of total, stubby, mushroom, and thin spines. Data are from n = 3 independent primary culture preparations. Each dot in the graphs represents the number of spines in a 100-µm-long dendrite. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, One-way ANOVA with Tukey’s post hoc test. G Immunofluorescence images of excitatory synapses in hippocampal cultures: WT, R6/2, R6/2 + GI; R6/2 + GI + ANA12. I Immunofluorescence images of excitatory synapses in hippocampal cultures: WT, zQ175, zQ175 + GI; zQ175 + GI + ANA12. Excitatory synapses in G and I were visualized by Bassoon/Homer1 immunostaining. Map2, pan neuronal marker. Upper panel scale bars: 50 μm; bottom panel scale bars: 10 μm. H , J Synapses quantification. Data are from n = 3 independent primary culture preparations. Each dot in the graphs represents the number of excitatory synapses in a 100-µm-long dendrite. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, One-way ANOVA with Tukey’s post-hoc test

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques: Inhibition, Transfection, Plasmid Preparation, Immunofluorescence, Immunostaining, Marker

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: Blocking active ADAM10 with GI254023X promotes LTP induction through the TrkB signaling pathway. A Experimental scheme of treatment of WT and R6/2 primary hippocampal neurons. The ADAM10 inhibitor GI254023X (GI, 1 µM) was administered from DIV6 until DIV14. The TrkB antagonist ANA12 (10 µM) was administered at DIV12 until DIV14. Chemical LTP was induced at DIV14 with 0.2 mM glycine for 15 min. For dendritic spine analyses hippocampal neurons were transfected at DIV5 with pcDNA3.1-mGreenLantern plasmid. B Representative traces of spontaneous EPSCs (sEPSCs) recorded at a holding potential of -70 mV in baseline condition and following chemical LTP-induction (cLTP) in primary hippocampal cell cultures obtained from WT and R6/2 mice. + GI and + ANA12 indicate the presence of these substances in culture medium and during electrophysiological recordings. C Graph comparing the amplitudes of sEPSCs in baseline condition and after cLTP induction in the different experimental conditions. Each dot corresponds to the value obtained from a single cell. Data are expressed as mean ± SEM and were analyzed by Two-way ANOVA with Bonferroni’s post hoc test. *P < 0.05, **P < 0.01. D Representative images of dendritic segments (mGreenLantern signal) and GluA1 immunostaining in basal condition and after cLTP induction. M, mushroom spines. Image crops of representative M spines were numbered from 1 to 6. Scale bars: 10 µm. E Quantification of mushroom spine density. Data are from n = 3 independent primary culture preparations. Each dot in the graph represents the number of mushroom spines in a 100-µm-long dendrite. Data are shown as % over the basal condition, which was set to 100, and are expressed as mean ± SEM. *P < 0.05, ***P < 0.001, unpaired t test. F Quantification of GluA1 signal. Data are from n = 3 independent primary culture preparations. Each dot in the graph represents GluA1 signal in a 100-µm-long dendrite. Data are expressed as mean ± SEM and were analyzed by Two-way ANOVA with Tukey’s post hoc test. P* < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. G Percentage of mushroom spines enriched in GluA1 in basal condition and after cLTP induction. Data are from n = 3–5 independent primary culture preparations. Each dot in the graph represents the number of mushroom spines in a 100-µm-long dendrite. Data are expressed as mean ± SEM and statistical analysis was performed by using Two-way ANOVA with Tukey’s post hoc test. P* < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Supplementary Table 1 and 2: detailed statistical outputs related to panel F and G

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques: Blocking Assay, Transfection, Plasmid Preparation, Immunostaining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

doi: 10.1007/s00018-024-05382-1

Figure Lengend Snippet: The ADAM10 and the BDNF/TrkB pathways at the HD hippocampal synapse. Defects in synaptic plasticity imply increased amounts of active ADAM10 in the HD hippocampus and downregulation of the BDNF/TrkB pathway. ADAM10 inhibition prevents the loss of long-lasting spines and enhances GluA1-AMPARs recruitment and LTP induction in mushroom spines, while also restoring BDNF and ERK signaling

Article Snippet: Separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Cat. No. 1704158) by means of the Trans-blot Turbo Transfer System (Bio-Rad) (High Molecular Weight protocol: 2.5 A constant; up to 25 V; 10 min), blocked with 5% nonfat milk (Bio-Rad, Cat. No. 1706404) in TBS1X and 0.1% Tween 20 (TBST) and incubated with rabbit polyclonal anti-ADAM10 antibody EPR5622 (1:1000 in TBST; Abcam, Cat. No. ab124695), mouse monoclonal anti-N-CAD antibody (1:1000 in TBST; Becton Dickinson Transduction Laboratories, Cat. No.610921), rabbit polyclonal anti-total-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9102), rabbit polyclonal anti-phospho-ERK1/2 antibody (1:2000 in TBST; Cell Signaling, Cat. No. 9101), mouse monoclonal anti-βIII-Tubulin antibody (1:1000 in TBST; Promega, Cat. No. G7121), and mouse monoclonal anti-α-Tubulin antibody (1:5000 in TBST; Millipore, Cat. No. T9026) at 4 °C overnight.

Techniques: Inhibition