rabbit anti adam10  (OriGene)

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

Journal: The Journal of biological chemistry

Article Title: Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43.

doi: 10.1016/j.jbc.2023.105272

Figure Lengend Snippet: Figure 3. Proteomic analyses suggest different intracellular origins of EV in the P1 and P2 fractions derived from HeLa cells exposed to bafilo- mycin A1 (Baf). HeLa cells were exposed to vehicle (DMSO) or 100 nM Baf for 24 h. Cell, P1, and P2 fractions in each treatment were prepared. A, immuno- EM images of vesicles in the P1 and P2 fractions derived from cells exposed to 100 nM Baf using the rabbit anti-C-terminal TDP-43 antibody. The image surrounded by a red brown square was magnified to compare vesicles between the P1 and P2 fractions. Scale bar represents 500 nm. B, a Venn diagram showing the distribution of proteins in the P1 and P2 fractions identified by LC–MS/MS analyses. C, representative immunoblots (ACTN4, ADAM10, p62, and α-tubulin) of the cell, P1, and P2 fractions are shown. D–F, densitometric data on (D) ACTN4, (E) ADAM10, and (F) p62 in the P1 and P2 fractions were calculated from immunoblotting results. In bar graphs, data are presented as means ± SD (N = 3) normalized against the P1 or P2 fraction derived from cells exposed to Baf. * indicates p < 0.05 by the two-tailed unpaired t test. DMSO, dimethyl sulfoxide; EV, extracellular vesicle; TDP-43, TAR DNA-binding protein 43 kDa.

Article Snippet: A monoclonal mouse anti-TDP-43 antibody (catalog no.: 60019-2-AP, Research Resource Identifier [RRID]: AB_2200520), polyclonal rabbit anti-C-terminal TDP-43 antibody (catalog no.: 12892-1-AP, RRID: AB_2200505), polyclonal rabbit anti-N-terminal TDP-43 antibody (catalog no.: 10782-2- AP, RRID: AB_615042), polyclonal rabbit anti-PGRN antibody (catalog no.: 18410-1-AP, RRID: AB_10598161), polyclonal rabbit anti-TSG101 antibody (catalog no.: 28283-1-AP, RRID: AB_2881104; catalog no.: 14497-1-AP, RRID: AB_2208090), polyclonal rabbit anti-ACTN4 antibody (catalog no.: 19096-1- AP, RRID: AB_10642150), polyclonal rabbit anti-ADAM10 antibody (catalog no.: 25900-1-AP, RRID: AB_2880291), and polyclonal rabbit anti-TFEB antibody (catalog no.: 13372-1-AP, RRID: AB_2919794) were purchased from ProteinTech; a monoclonal mouse anti-α-tubulin antibody (catalog no.: 70- 102) and polyclonal rabbit anti-GFP antibody (catalog no.: 60-011) from BioAcademia; a monoclonal mouse anti-LC3 antibody (catalog no.: M186-3), normal rabbit immunoglobulin G (IgG) (catalog no.: PM035), polyclonal rabbit antiSyntaxin-17 (human) antibody (catalog no.: PM076), and polyclonal rabbit anti-ATG16L1 antibody (catalog no.: PM040, RRID: AB_1278757) from MBL; a monoclonal mouse anti-p62 antibody (catalog no.: 610832, RRID: AB_398151) from BD control siRNA were calculated from three independent experiments including a 48 h were stained with AcidiFluor ORANGE, and the acidity of lysosomes was c and photographed at a bright field are shown.

Techniques: Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Two Tailed Test, Binding Assay

Journal: Neural Regeneration Research

Article Title: ADAM10 facilitates rapid neural stem cell cycling and proper positioning within the subventricular zone niche via JAMC/RAP1Gap signaling

doi: 10.4103/1673-5374.339007

Figure Lengend Snippet: Primary and secondary antibodies used in this study

Article Snippet: Rabbit polyclonal anti-ADAM10 , Abcam, ab1997 , AB_302747.

Techniques: