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NewEast Biosciences mouse anti rhoa gtp monoclonal antibody
Enhanced VEGFA and Rho signaling pathway in LSECs from Adamts18 −/− mice (A) Immunofluorescence staining for CD14 in primary LSECs from 2-week-old Adamts18 +/+ and Adamts18 −/− mice. Scale bar, 50 μm. (B) Representative western blots for VEGFA, pSrc, Src, <t>RhoA-GTP,</t> RhoA, pYAP, YAP, pMLC, and MLC in primary LSECs. (C) The relative quantity of VEGFA is normalized to that of GAPDH and phosphorylated proteins is normalized to that of corresponding total proteins. Data are expressed as mean ± SD ( n = 3). (D) Representative immunofluorescence staining for RhoA-GTP, pYAP, and pMLC in primary LSECs. Scale bar, 10 μm. (E) Quantification of the fluorescence intensity in panel D by ImageJ. Each LSEC slide was analyzed for 5 fields. Data are represented as mean ± SD ( n = 3). Each dot or square represents one individual. Ns, no significance, ∗ p < 0.05, ∗∗ p < 0.01; two-tailed Student’s t test.
Mouse Anti Rhoa Gtp Monoclonal Antibody, supplied by NewEast Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "ADAMTS18-fibronectin interaction regulates the morphology of liver sinusoidal endothelial cells"

Article Title: ADAMTS18-fibronectin interaction regulates the morphology of liver sinusoidal endothelial cells

Journal: iScience

doi: 10.1016/j.isci.2024.110273

Enhanced VEGFA and Rho signaling pathway in LSECs from Adamts18 −/− mice (A) Immunofluorescence staining for CD14 in primary LSECs from 2-week-old Adamts18 +/+ and Adamts18 −/− mice. Scale bar, 50 μm. (B) Representative western blots for VEGFA, pSrc, Src, RhoA-GTP, RhoA, pYAP, YAP, pMLC, and MLC in primary LSECs. (C) The relative quantity of VEGFA is normalized to that of GAPDH and phosphorylated proteins is normalized to that of corresponding total proteins. Data are expressed as mean ± SD ( n = 3). (D) Representative immunofluorescence staining for RhoA-GTP, pYAP, and pMLC in primary LSECs. Scale bar, 10 μm. (E) Quantification of the fluorescence intensity in panel D by ImageJ. Each LSEC slide was analyzed for 5 fields. Data are represented as mean ± SD ( n = 3). Each dot or square represents one individual. Ns, no significance, ∗ p < 0.05, ∗∗ p < 0.01; two-tailed Student’s t test.
Figure Legend Snippet: Enhanced VEGFA and Rho signaling pathway in LSECs from Adamts18 −/− mice (A) Immunofluorescence staining for CD14 in primary LSECs from 2-week-old Adamts18 +/+ and Adamts18 −/− mice. Scale bar, 50 μm. (B) Representative western blots for VEGFA, pSrc, Src, RhoA-GTP, RhoA, pYAP, YAP, pMLC, and MLC in primary LSECs. (C) The relative quantity of VEGFA is normalized to that of GAPDH and phosphorylated proteins is normalized to that of corresponding total proteins. Data are expressed as mean ± SD ( n = 3). (D) Representative immunofluorescence staining for RhoA-GTP, pYAP, and pMLC in primary LSECs. Scale bar, 10 μm. (E) Quantification of the fluorescence intensity in panel D by ImageJ. Each LSEC slide was analyzed for 5 fields. Data are represented as mean ± SD ( n = 3). Each dot or square represents one individual. Ns, no significance, ∗ p < 0.05, ∗∗ p < 0.01; two-tailed Student’s t test.

Techniques Used: Immunofluorescence, Staining, Western Blot, Fluorescence, Two Tailed Test


Figure Legend Snippet:

Techniques Used: Recombinant, RNAscope, SYBR Green Assay, Modification, Staining, Peroxidation Assay, Immunoprecipitation, Activity Assay, Plasmid Preparation, Software



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NewEast Biosciences mouse anti rhoa gtp monoclonal antibody
Enhanced VEGFA and Rho signaling pathway in LSECs from Adamts18 −/− mice (A) Immunofluorescence staining for CD14 in primary LSECs from 2-week-old Adamts18 +/+ and Adamts18 −/− mice. Scale bar, 50 μm. (B) Representative western blots for VEGFA, pSrc, Src, <t>RhoA-GTP,</t> RhoA, pYAP, YAP, pMLC, and MLC in primary LSECs. (C) The relative quantity of VEGFA is normalized to that of GAPDH and phosphorylated proteins is normalized to that of corresponding total proteins. Data are expressed as mean ± SD ( n = 3). (D) Representative immunofluorescence staining for RhoA-GTP, pYAP, and pMLC in primary LSECs. Scale bar, 10 μm. (E) Quantification of the fluorescence intensity in panel D by ImageJ. Each LSEC slide was analyzed for 5 fields. Data are represented as mean ± SD ( n = 3). Each dot or square represents one individual. Ns, no significance, ∗ p < 0.05, ∗∗ p < 0.01; two-tailed Student’s t test.
Mouse Anti Rhoa Gtp Monoclonal Antibody, supplied by NewEast Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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NewEast Biosciences mouse monoclonal anti rhoa gtp

Mouse Monoclonal Anti Rhoa Gtp, supplied by NewEast Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Cytoskeleton Inc anti gtp rhoa
ARHGAP35 regulates cytoskeletal reorganization and epithelial-to-mesenchymal transition by targeting <t>RhoA</t> and E-cadherin, respectively. a-b, GC cell cytoskeleton staining was performed using phalloidin. c, The protein expression levels of RhoA and <t>GTP-RhoA</t> were detected by western blot analysis. d, The protein expression levels of e-cadherin were determined in ARHGAP35-depleted or -overexpressing GC cells using western blot analysis. e-f: The expression of the epithelial marker, e-cadherin, in GC cells was assessed by immunofluorescence staining. ARHGAP35, Rho GTPase‑activating protein 35; GC, gastric cancer.
Anti Gtp Rhoa, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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NewEast Biosciences mouse monoclonal anti rhoa gtp antibody
The NPY/Y5R pathway activates <t>RhoA.</t> (A) Immunocytochemistry with anti-Y5R (green) and <t>anti-RhoA-GTP</t> (red) antibodies in CHO-K1/Y5R-EGFP cells. (B) RhoA pull-down assay in CHO-K1/Y5R-EGFP cells treated with NPY (10 −7 M). (C) RhoA pull-down assay in SK-N-AS NB cells treated with NPY (10 −10 -10 −7 M). (D) Quantitative analysis of RhoA activity measured by a pull-down assay in SK-N-AS cells treated with NPY at a concentration of 10 −8 M. (C,D) * p < 0.05 by t -test; mean ± SEM from three independent experiments.
Mouse Monoclonal Anti Rhoa Gtp Antibody, supplied by NewEast Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Synaptic proteins, neuronal markers and Tau kinase activity in pR75KO at 6 months. ( A ) Protein blots of kinases involved in Tau phosphorylation, GSK3β, <t>RhoA</t> and Cdk5 activators, p35 and p25 proteins in the forebrain of Wt, p75KO, pR5, and pR75KO mice. Protein band intensity quantification of inactive GSK3:GSK3β pS9 normalised with total GSK3β ( B ), Cdk5 activators, p25/p35 ratio ( C ), and active <t>RhoA-GTP</t> normalised with total RhoA ( D ). All band intensities showing B-D are expressed as fold change relative to Wt. F) Protein blots of post-synaptic protein, PSD-95 and pre-synaptic proteins, SNAP25 and VAMP2, tyrosine hydroxylase (TH) and choline acetyl transferase (ChAT). Protein band intensity quantification of PSD-95 ( E ), SNAP25 ( G ), VAMP2 ( H ), choline acetyl transferase (ChAT) ( I ), and tyrosine hydroxylase (TH) ( J ) normalised with total β-actin of respective blot and expressed as fold change relative to Wt. Data are represented as the mean ± SEM, n=6. Statistical comparisons were performed using one-way ANOVA and Tukey’s test. Statistical significance: *P<0.05, **P<0.01 , ***P<0.001, ****P<0.0001 .
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Synaptic proteins, neuronal markers and Tau kinase activity in pR75KO at 6 months. ( A ) Protein blots of kinases involved in Tau phosphorylation, GSK3β, <t>RhoA</t> and Cdk5 activators, p35 and p25 proteins in the forebrain of Wt, p75KO, pR5, and pR75KO mice. Protein band intensity quantification of inactive GSK3:GSK3β pS9 normalised with total GSK3β ( B ), Cdk5 activators, p25/p35 ratio ( C ), and active <t>RhoA-GTP</t> normalised with total RhoA ( D ). All band intensities showing B-D are expressed as fold change relative to Wt. F) Protein blots of post-synaptic protein, PSD-95 and pre-synaptic proteins, SNAP25 and VAMP2, tyrosine hydroxylase (TH) and choline acetyl transferase (ChAT). Protein band intensity quantification of PSD-95 ( E ), SNAP25 ( G ), VAMP2 ( H ), choline acetyl transferase (ChAT) ( I ), and tyrosine hydroxylase (TH) ( J ) normalised with total β-actin of respective blot and expressed as fold change relative to Wt. Data are represented as the mean ± SEM, n=6. Statistical comparisons were performed using one-way ANOVA and Tukey’s test. Statistical significance: *P<0.05, **P<0.01 , ***P<0.001, ****P<0.0001 .
Mouse Monoclonal Anti Rhoa Gtp, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Enhanced VEGFA and Rho signaling pathway in LSECs from Adamts18 −/− mice (A) Immunofluorescence staining for CD14 in primary LSECs from 2-week-old Adamts18 +/+ and Adamts18 −/− mice. Scale bar, 50 μm. (B) Representative western blots for VEGFA, pSrc, Src, RhoA-GTP, RhoA, pYAP, YAP, pMLC, and MLC in primary LSECs. (C) The relative quantity of VEGFA is normalized to that of GAPDH and phosphorylated proteins is normalized to that of corresponding total proteins. Data are expressed as mean ± SD ( n = 3). (D) Representative immunofluorescence staining for RhoA-GTP, pYAP, and pMLC in primary LSECs. Scale bar, 10 μm. (E) Quantification of the fluorescence intensity in panel D by ImageJ. Each LSEC slide was analyzed for 5 fields. Data are represented as mean ± SD ( n = 3). Each dot or square represents one individual. Ns, no significance, ∗ p < 0.05, ∗∗ p < 0.01; two-tailed Student’s t test.

Journal: iScience

Article Title: ADAMTS18-fibronectin interaction regulates the morphology of liver sinusoidal endothelial cells

doi: 10.1016/j.isci.2024.110273

Figure Lengend Snippet: Enhanced VEGFA and Rho signaling pathway in LSECs from Adamts18 −/− mice (A) Immunofluorescence staining for CD14 in primary LSECs from 2-week-old Adamts18 +/+ and Adamts18 −/− mice. Scale bar, 50 μm. (B) Representative western blots for VEGFA, pSrc, Src, RhoA-GTP, RhoA, pYAP, YAP, pMLC, and MLC in primary LSECs. (C) The relative quantity of VEGFA is normalized to that of GAPDH and phosphorylated proteins is normalized to that of corresponding total proteins. Data are expressed as mean ± SD ( n = 3). (D) Representative immunofluorescence staining for RhoA-GTP, pYAP, and pMLC in primary LSECs. Scale bar, 10 μm. (E) Quantification of the fluorescence intensity in panel D by ImageJ. Each LSEC slide was analyzed for 5 fields. Data are represented as mean ± SD ( n = 3). Each dot or square represents one individual. Ns, no significance, ∗ p < 0.05, ∗∗ p < 0.01; two-tailed Student’s t test.

Article Snippet: Mouse Anti-RhoA-GTP Monoclonal Antibody , NewEast Biosciences , Cat# 26904, RRID: AB_1961799.

Techniques: Immunofluorescence, Staining, Western Blot, Fluorescence, Two Tailed Test

Journal: iScience

Article Title: ADAMTS18-fibronectin interaction regulates the morphology of liver sinusoidal endothelial cells

doi: 10.1016/j.isci.2024.110273

Figure Lengend Snippet:

Article Snippet: Mouse Anti-RhoA-GTP Monoclonal Antibody , NewEast Biosciences , Cat# 26904, RRID: AB_1961799.

Techniques: Recombinant, RNAscope, SYBR Green Assay, Modification, Staining, Peroxidation Assay, Immunoprecipitation, Activity Assay, Plasmid Preparation, Software

Journal: Cell Reports Medicine

Article Title: Alarmin S100A8 imparts chemoresistance of esophageal cancer by reprogramming cancer-associated fibroblasts

doi: 10.1016/j.xcrm.2024.101576

Figure Lengend Snippet:

Article Snippet: Mouse Monoclonal anti-RhoA-GTP , NewEast Biosciences , Cat# 26904; RRID: AB_1961799.

Techniques: Virus, shRNA, Over Expression, Recombinant, Purification, Staining, Enzyme-linked Immunosorbent Assay, Sonication, Chromatin Immunoprecipitation, Software

ARHGAP35 regulates cytoskeletal reorganization and epithelial-to-mesenchymal transition by targeting RhoA and E-cadherin, respectively. a-b, GC cell cytoskeleton staining was performed using phalloidin. c, The protein expression levels of RhoA and GTP-RhoA were detected by western blot analysis. d, The protein expression levels of e-cadherin were determined in ARHGAP35-depleted or -overexpressing GC cells using western blot analysis. e-f: The expression of the epithelial marker, e-cadherin, in GC cells was assessed by immunofluorescence staining. ARHGAP35, Rho GTPase‑activating protein 35; GC, gastric cancer.

Journal: Bioengineered

Article Title: Rho GTPase-activating protein 35 suppresses gastric cancer metastasis by regulating cytoskeleton reorganization and epithelial-to-mesenchymal transition

doi: 10.1080/21655979.2022.2092677

Figure Lengend Snippet: ARHGAP35 regulates cytoskeletal reorganization and epithelial-to-mesenchymal transition by targeting RhoA and E-cadherin, respectively. a-b, GC cell cytoskeleton staining was performed using phalloidin. c, The protein expression levels of RhoA and GTP-RhoA were detected by western blot analysis. d, The protein expression levels of e-cadherin were determined in ARHGAP35-depleted or -overexpressing GC cells using western blot analysis. e-f: The expression of the epithelial marker, e-cadherin, in GC cells was assessed by immunofluorescence staining. ARHGAP35, Rho GTPase‑activating protein 35; GC, gastric cancer.

Article Snippet: The antibodies used were as follows: Anti-ARHGAP35 (cat. no. 2860), anti-E-cadherin (cat. no. 3195S; both from Cell Signaling Technology, Inc.), anti-RhoA (cat. no. ab187027; Abcam), anti-GTP-RhoA (cat. no. ARH05; Cytoskeleton, Inc.) and anti-β-actin (cat. no. A2228; clone AC-74; MilliporeSigma).

Techniques: Staining, Expressing, Western Blot, Marker, Immunofluorescence

The NPY/Y5R pathway activates RhoA. (A) Immunocytochemistry with anti-Y5R (green) and anti-RhoA-GTP (red) antibodies in CHO-K1/Y5R-EGFP cells. (B) RhoA pull-down assay in CHO-K1/Y5R-EGFP cells treated with NPY (10 −7 M). (C) RhoA pull-down assay in SK-N-AS NB cells treated with NPY (10 −10 -10 −7 M). (D) Quantitative analysis of RhoA activity measured by a pull-down assay in SK-N-AS cells treated with NPY at a concentration of 10 −8 M. (C,D) * p < 0.05 by t -test; mean ± SEM from three independent experiments.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Neuropeptide Y/Y5 Receptor Pathway Stimulates Neuroblastoma Cell Motility Through RhoA Activation

doi: 10.3389/fcell.2020.627090

Figure Lengend Snippet: The NPY/Y5R pathway activates RhoA. (A) Immunocytochemistry with anti-Y5R (green) and anti-RhoA-GTP (red) antibodies in CHO-K1/Y5R-EGFP cells. (B) RhoA pull-down assay in CHO-K1/Y5R-EGFP cells treated with NPY (10 −7 M). (C) RhoA pull-down assay in SK-N-AS NB cells treated with NPY (10 −10 -10 −7 M). (D) Quantitative analysis of RhoA activity measured by a pull-down assay in SK-N-AS cells treated with NPY at a concentration of 10 −8 M. (C,D) * p < 0.05 by t -test; mean ± SEM from three independent experiments.

Article Snippet: Then, the cells were permeabilized and stained with mouse monoclonal anti-RhoA-GTP antibody (1:100; NewEast Biosciences, King of Prussia, PA; cat # NE-26904) overnight at 4°C, followed by incubation with AlexaFluor 594-conjugated donkey anti-mouse antibody (1:1,000, Invitrogen; cat # A-21203) for 1 h at RT.

Techniques: Immunocytochemistry, Pull Down Assay, Activity Assay, Concentration Assay

In situ proximity ligation assay (PLA) reveals interactions between Y5R and RhoA-GTP. PLA performed in SK-N-AS cells with anti-Y5R and anti-RhoA-GTP antibodies. (A) Representative confocal microscopy images of PLA in SK-N-AS cells treated with NPY (10 −8 M) for 5, 10, and 20 min. Red dots represent interactions between Y5R and RhoA-GTP. The graph depicts quantification of fluorescence intensities for the total PLA signal or the signal located inside of the cell colonies and near their outer membranes in SK-N-AS cells treated with NPY. (B) PLA (red) and phalloidin staining (green) in SK-N-AS cells treated with NPY (10 −8 M) for 20 min, with or without Y5R antagonist (10 −6 M) pre-treatment for 30 min. White arrows indicate leading edges of the neuroblastoma (NB) cells. The graph represents a quantification of fluorescence intensity of the PLA signal—total or located inside of cell colonies and near their outer membranes in SK-N-AS cells treated with NPY and Y5R antagonist as above. (A,B) * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. non-treated control or **** p < 0.0001 as indicated, by one-way ANOVA followed by Tukey's test; mean ± SEM, n = 11–21 per condition. (C) Co-localization of the PLA signal (red) with F-actin (green) on the outer membranes of cells within the colony with migratory phenotype. (D) PLA signal in the area rich in F-actin near the outer plasma membrane. (E) Localization of the PLA signal along F-actin fibers.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Neuropeptide Y/Y5 Receptor Pathway Stimulates Neuroblastoma Cell Motility Through RhoA Activation

doi: 10.3389/fcell.2020.627090

Figure Lengend Snippet: In situ proximity ligation assay (PLA) reveals interactions between Y5R and RhoA-GTP. PLA performed in SK-N-AS cells with anti-Y5R and anti-RhoA-GTP antibodies. (A) Representative confocal microscopy images of PLA in SK-N-AS cells treated with NPY (10 −8 M) for 5, 10, and 20 min. Red dots represent interactions between Y5R and RhoA-GTP. The graph depicts quantification of fluorescence intensities for the total PLA signal or the signal located inside of the cell colonies and near their outer membranes in SK-N-AS cells treated with NPY. (B) PLA (red) and phalloidin staining (green) in SK-N-AS cells treated with NPY (10 −8 M) for 20 min, with or without Y5R antagonist (10 −6 M) pre-treatment for 30 min. White arrows indicate leading edges of the neuroblastoma (NB) cells. The graph represents a quantification of fluorescence intensity of the PLA signal—total or located inside of cell colonies and near their outer membranes in SK-N-AS cells treated with NPY and Y5R antagonist as above. (A,B) * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. non-treated control or **** p < 0.0001 as indicated, by one-way ANOVA followed by Tukey's test; mean ± SEM, n = 11–21 per condition. (C) Co-localization of the PLA signal (red) with F-actin (green) on the outer membranes of cells within the colony with migratory phenotype. (D) PLA signal in the area rich in F-actin near the outer plasma membrane. (E) Localization of the PLA signal along F-actin fibers.

Article Snippet: Then, the cells were permeabilized and stained with mouse monoclonal anti-RhoA-GTP antibody (1:100; NewEast Biosciences, King of Prussia, PA; cat # NE-26904) overnight at 4°C, followed by incubation with AlexaFluor 594-conjugated donkey anti-mouse antibody (1:1,000, Invitrogen; cat # A-21203) for 1 h at RT.

Techniques: In Situ, Proximity Ligation Assay, Confocal Microscopy, Fluorescence, Staining

Synaptic proteins, neuronal markers and Tau kinase activity in pR75KO at 6 months. ( A ) Protein blots of kinases involved in Tau phosphorylation, GSK3β, RhoA and Cdk5 activators, p35 and p25 proteins in the forebrain of Wt, p75KO, pR5, and pR75KO mice. Protein band intensity quantification of inactive GSK3:GSK3β pS9 normalised with total GSK3β ( B ), Cdk5 activators, p25/p35 ratio ( C ), and active RhoA-GTP normalised with total RhoA ( D ). All band intensities showing B-D are expressed as fold change relative to Wt. F) Protein blots of post-synaptic protein, PSD-95 and pre-synaptic proteins, SNAP25 and VAMP2, tyrosine hydroxylase (TH) and choline acetyl transferase (ChAT). Protein band intensity quantification of PSD-95 ( E ), SNAP25 ( G ), VAMP2 ( H ), choline acetyl transferase (ChAT) ( I ), and tyrosine hydroxylase (TH) ( J ) normalised with total β-actin of respective blot and expressed as fold change relative to Wt. Data are represented as the mean ± SEM, n=6. Statistical comparisons were performed using one-way ANOVA and Tukey’s test. Statistical significance: *P<0.05, **P<0.01 , ***P<0.001, ****P<0.0001 .

Journal: Aging (Albany NY)

Article Title: Knockout of p75 neurotrophin receptor attenuates the hyperphosphorylation of Tau in pR5 mouse model

doi: 10.18632/aging.102202

Figure Lengend Snippet: Synaptic proteins, neuronal markers and Tau kinase activity in pR75KO at 6 months. ( A ) Protein blots of kinases involved in Tau phosphorylation, GSK3β, RhoA and Cdk5 activators, p35 and p25 proteins in the forebrain of Wt, p75KO, pR5, and pR75KO mice. Protein band intensity quantification of inactive GSK3:GSK3β pS9 normalised with total GSK3β ( B ), Cdk5 activators, p25/p35 ratio ( C ), and active RhoA-GTP normalised with total RhoA ( D ). All band intensities showing B-D are expressed as fold change relative to Wt. F) Protein blots of post-synaptic protein, PSD-95 and pre-synaptic proteins, SNAP25 and VAMP2, tyrosine hydroxylase (TH) and choline acetyl transferase (ChAT). Protein band intensity quantification of PSD-95 ( E ), SNAP25 ( G ), VAMP2 ( H ), choline acetyl transferase (ChAT) ( I ), and tyrosine hydroxylase (TH) ( J ) normalised with total β-actin of respective blot and expressed as fold change relative to Wt. Data are represented as the mean ± SEM, n=6. Statistical comparisons were performed using one-way ANOVA and Tukey’s test. Statistical significance: *P<0.05, **P<0.01 , ***P<0.001, ****P<0.0001 .

Article Snippet: MN1020, CiteAb, UK); sheep anti-human Tau (Antibody Technology Australia, Australia); cleaved caspase-3, rabbit anti-GSK3α/β (Ser21/9), rabbit anti-GSK3β pS9, rabbit anti-p25/35, anti-phosphorylated PKA T197, and anti-total PKA C-α (Cell Signalling Technology, QLD, Australia); rabbit affinity purified anti-ChAT was from Dr . John Oliver (Centre for Neuroscience, Department of Human Physiology, Flinders University); mouse anti-tyrosine hydroxylase (TH) (Sigma-Aldrich, St Louis, MO, US) anti-neuronal nuclei antigen (anti-NeuN) (Merck Millipore, VIC, Australia); rabbit anti-glial fibrillary acidic protein (anti-GFAP) (DAKO, Denmark); rabbit anti-vesicle-associated membrane protein 2 (VAMP2) and rabbit anti-synaptosomal-associated protein 25 (SNAP25) (OSS00035W, Osenses, Australia); anti-postsynaptic density protein 95 (PSD-95) (Sigma-Aldrich, St Louis, MO, US);and mouse monoclonal anti-RhoA-GTP and rabbit anti-total RhoA (New-East Biosciences, Malvern, Pennsylvania, US).

Techniques: Activity Assay

Knock out of p75 NTR attenuated Tau hyperposphorylation and the elevated Tau kinases and caspase-3 activities observed in pR5 mice with P301L Tau at 9 months. ( A ) Protein blots of phosphorylated and non-phosphorylated human Tau in in the forebrain of Wt, p75KO, pR5, and pR75KO mice. ( B ) Protein band intensity quantification of phosphorylated human Tau at sites S262, S396 and S202/T205 (AT8) normalised to the total human Tau and expressed as fold change relative to pR5. Protein band intensity quantification of total human Tau and pTau detected by HT7 ( C ), total mouse Tau detected by Tau5 ( D ), and total human Tau detected by sheep-anti human Tau ( E ) normalised to β-actin and expressed as fold change relative to Wt. ( F ) Protein blots of kinases involved in Tau phosphorylation, GSK3, RhoA and Cdk5-activators, p25 and p35 proteins in the forebrain of Wt, p75KO, pR5, and pR75KO mice; of cleaved caspase-3; and of post-synaptic protein, PSD-95 and pre-synaptic proteins, SNAP25 and VAMP2, GFAP, TH ChAT. Protein band intensity quantification of inactive GSK3: GSK3β pS9 normalised with total GSK3β ( G ), active RhoA-GTP normalised with total RhoA ( H ), and Cdk5 activators, p25/p35 ratio ( I ). All band intensities showing ( G – I ) are expressed as fold change relative to Wt. Protein band intensity quantification of cleaved caspase-3 levels ( J ), PSD-95 ( K ), SNAP25 ( L ), VAMP2 ( M ), TH ( N ), GFAP ( O ), ChAT ( P ) normalized with their respective β-actin and expressed as fold change relative to Wt. Data are represented as the mean ± SEM, n=3. Statistical comparisons were performed using one-way ANOVA and Tukey’s test. For human pTau, two-tailed unpaired t-test was used to compare pR5 and pR75KO mice Statistical significance: *P<0.05, **P<0.01 , ***P<0.001, ****P<0.0001 .

Journal: Aging (Albany NY)

Article Title: Knockout of p75 neurotrophin receptor attenuates the hyperphosphorylation of Tau in pR5 mouse model

doi: 10.18632/aging.102202

Figure Lengend Snippet: Knock out of p75 NTR attenuated Tau hyperposphorylation and the elevated Tau kinases and caspase-3 activities observed in pR5 mice with P301L Tau at 9 months. ( A ) Protein blots of phosphorylated and non-phosphorylated human Tau in in the forebrain of Wt, p75KO, pR5, and pR75KO mice. ( B ) Protein band intensity quantification of phosphorylated human Tau at sites S262, S396 and S202/T205 (AT8) normalised to the total human Tau and expressed as fold change relative to pR5. Protein band intensity quantification of total human Tau and pTau detected by HT7 ( C ), total mouse Tau detected by Tau5 ( D ), and total human Tau detected by sheep-anti human Tau ( E ) normalised to β-actin and expressed as fold change relative to Wt. ( F ) Protein blots of kinases involved in Tau phosphorylation, GSK3, RhoA and Cdk5-activators, p25 and p35 proteins in the forebrain of Wt, p75KO, pR5, and pR75KO mice; of cleaved caspase-3; and of post-synaptic protein, PSD-95 and pre-synaptic proteins, SNAP25 and VAMP2, GFAP, TH ChAT. Protein band intensity quantification of inactive GSK3: GSK3β pS9 normalised with total GSK3β ( G ), active RhoA-GTP normalised with total RhoA ( H ), and Cdk5 activators, p25/p35 ratio ( I ). All band intensities showing ( G – I ) are expressed as fold change relative to Wt. Protein band intensity quantification of cleaved caspase-3 levels ( J ), PSD-95 ( K ), SNAP25 ( L ), VAMP2 ( M ), TH ( N ), GFAP ( O ), ChAT ( P ) normalized with their respective β-actin and expressed as fold change relative to Wt. Data are represented as the mean ± SEM, n=3. Statistical comparisons were performed using one-way ANOVA and Tukey’s test. For human pTau, two-tailed unpaired t-test was used to compare pR5 and pR75KO mice Statistical significance: *P<0.05, **P<0.01 , ***P<0.001, ****P<0.0001 .

Article Snippet: MN1020, CiteAb, UK); sheep anti-human Tau (Antibody Technology Australia, Australia); cleaved caspase-3, rabbit anti-GSK3α/β (Ser21/9), rabbit anti-GSK3β pS9, rabbit anti-p25/35, anti-phosphorylated PKA T197, and anti-total PKA C-α (Cell Signalling Technology, QLD, Australia); rabbit affinity purified anti-ChAT was from Dr . John Oliver (Centre for Neuroscience, Department of Human Physiology, Flinders University); mouse anti-tyrosine hydroxylase (TH) (Sigma-Aldrich, St Louis, MO, US) anti-neuronal nuclei antigen (anti-NeuN) (Merck Millipore, VIC, Australia); rabbit anti-glial fibrillary acidic protein (anti-GFAP) (DAKO, Denmark); rabbit anti-vesicle-associated membrane protein 2 (VAMP2) and rabbit anti-synaptosomal-associated protein 25 (SNAP25) (OSS00035W, Osenses, Australia); anti-postsynaptic density protein 95 (PSD-95) (Sigma-Aldrich, St Louis, MO, US);and mouse monoclonal anti-RhoA-GTP and rabbit anti-total RhoA (New-East Biosciences, Malvern, Pennsylvania, US).

Techniques: Knock-Out, Two Tailed Test

Synaptic proteins, neuronal markers and Tau kinase activity in pR75KO at 6 months. ( A ) Protein blots of kinases involved in Tau phosphorylation, GSK3β, RhoA and Cdk5 activators, p35 and p25 proteins in the forebrain of Wt, p75KO, pR5, and pR75KO mice. Protein band intensity quantification of inactive GSK3:GSK3β pS9 normalised with total GSK3β ( B ), Cdk5 activators, p25/p35 ratio ( C ), and active RhoA-GTP normalised with total RhoA ( D ). All band intensities showing B-D are expressed as fold change relative to Wt. F) Protein blots of post-synaptic protein, PSD-95 and pre-synaptic proteins, SNAP25 and VAMP2, tyrosine hydroxylase (TH) and choline acetyl transferase (ChAT). Protein band intensity quantification of PSD-95 ( E ), SNAP25 ( G ), VAMP2 ( H ), choline acetyl transferase (ChAT) ( I ), and tyrosine hydroxylase (TH) ( J ) normalised with total β-actin of respective blot and expressed as fold change relative to Wt. Data are represented as the mean ± SEM, n=6. Statistical comparisons were performed using one-way ANOVA and Tukey’s test. Statistical significance: *P<0.05, **P<0.01 , ***P<0.001, ****P<0.0001 .

Journal: Aging (Albany NY)

Article Title: Knockout of p75 neurotrophin receptor attenuates the hyperphosphorylation of Tau in pR5 mouse model

doi: 10.18632/aging.102202

Figure Lengend Snippet: Synaptic proteins, neuronal markers and Tau kinase activity in pR75KO at 6 months. ( A ) Protein blots of kinases involved in Tau phosphorylation, GSK3β, RhoA and Cdk5 activators, p35 and p25 proteins in the forebrain of Wt, p75KO, pR5, and pR75KO mice. Protein band intensity quantification of inactive GSK3:GSK3β pS9 normalised with total GSK3β ( B ), Cdk5 activators, p25/p35 ratio ( C ), and active RhoA-GTP normalised with total RhoA ( D ). All band intensities showing B-D are expressed as fold change relative to Wt. F) Protein blots of post-synaptic protein, PSD-95 and pre-synaptic proteins, SNAP25 and VAMP2, tyrosine hydroxylase (TH) and choline acetyl transferase (ChAT). Protein band intensity quantification of PSD-95 ( E ), SNAP25 ( G ), VAMP2 ( H ), choline acetyl transferase (ChAT) ( I ), and tyrosine hydroxylase (TH) ( J ) normalised with total β-actin of respective blot and expressed as fold change relative to Wt. Data are represented as the mean ± SEM, n=6. Statistical comparisons were performed using one-way ANOVA and Tukey’s test. Statistical significance: *P<0.05, **P<0.01 , ***P<0.001, ****P<0.0001 .

Article Snippet: MN1020, CiteAb, UK); sheep anti-human Tau (Antibody Technology Australia, Australia); cleaved caspase-3, rabbit anti-GSK3α/β (Ser21/9), rabbit anti-GSK3β pS9, rabbit anti-p25/35, anti-phosphorylated PKA T197, and anti-total PKA C-α (Cell Signalling Technology, QLD, Australia); rabbit affinity purified anti-ChAT was from Dr . John Oliver (Centre for Neuroscience, Department of Human Physiology, Flinders University); mouse anti-tyrosine hydroxylase (TH) (Sigma-Aldrich, St Louis, MO, US) anti-neuronal nuclei antigen (anti-NeuN) (Merck Millipore, VIC, Australia); rabbit anti-glial fibrillary acidic protein (anti-GFAP) (DAKO, Denmark); rabbit anti-vesicle-associated membrane protein 2 (VAMP2) and rabbit anti-synaptosomal-associated protein 25 (SNAP25) (OSS00035W, Osenses, Australia); anti-postsynaptic density protein 95 (PSD-95) (Sigma-Aldrich, St Louis, MO, US);and mouse monoclonal anti-RhoA-GTP and rabbit anti-total RhoA (New-East Biosciences, Malvern, Pennsylvania, US).

Techniques: Activity Assay

Knock out of p75 NTR attenuated Tau hyperposphorylation and the elevated Tau kinases and caspase-3 activities observed in pR5 mice with P301L Tau at 9 months. ( A ) Protein blots of phosphorylated and non-phosphorylated human Tau in in the forebrain of Wt, p75KO, pR5, and pR75KO mice. ( B ) Protein band intensity quantification of phosphorylated human Tau at sites S262, S396 and S202/T205 (AT8) normalised to the total human Tau and expressed as fold change relative to pR5. Protein band intensity quantification of total human Tau and pTau detected by HT7 ( C ), total mouse Tau detected by Tau5 ( D ), and total human Tau detected by sheep-anti human Tau ( E ) normalised to β-actin and expressed as fold change relative to Wt. ( F ) Protein blots of kinases involved in Tau phosphorylation, GSK3, RhoA and Cdk5-activators, p25 and p35 proteins in the forebrain of Wt, p75KO, pR5, and pR75KO mice; of cleaved caspase-3; and of post-synaptic protein, PSD-95 and pre-synaptic proteins, SNAP25 and VAMP2, GFAP, TH ChAT. Protein band intensity quantification of inactive GSK3: GSK3β pS9 normalised with total GSK3β ( G ), active RhoA-GTP normalised with total RhoA ( H ), and Cdk5 activators, p25/p35 ratio ( I ). All band intensities showing ( G – I ) are expressed as fold change relative to Wt. Protein band intensity quantification of cleaved caspase-3 levels ( J ), PSD-95 ( K ), SNAP25 ( L ), VAMP2 ( M ), TH ( N ), GFAP ( O ), ChAT ( P ) normalized with their respective β-actin and expressed as fold change relative to Wt. Data are represented as the mean ± SEM, n=3. Statistical comparisons were performed using one-way ANOVA and Tukey’s test. For human pTau, two-tailed unpaired t-test was used to compare pR5 and pR75KO mice Statistical significance: *P<0.05, **P<0.01 , ***P<0.001, ****P<0.0001 .

Journal: Aging (Albany NY)

Article Title: Knockout of p75 neurotrophin receptor attenuates the hyperphosphorylation of Tau in pR5 mouse model

doi: 10.18632/aging.102202

Figure Lengend Snippet: Knock out of p75 NTR attenuated Tau hyperposphorylation and the elevated Tau kinases and caspase-3 activities observed in pR5 mice with P301L Tau at 9 months. ( A ) Protein blots of phosphorylated and non-phosphorylated human Tau in in the forebrain of Wt, p75KO, pR5, and pR75KO mice. ( B ) Protein band intensity quantification of phosphorylated human Tau at sites S262, S396 and S202/T205 (AT8) normalised to the total human Tau and expressed as fold change relative to pR5. Protein band intensity quantification of total human Tau and pTau detected by HT7 ( C ), total mouse Tau detected by Tau5 ( D ), and total human Tau detected by sheep-anti human Tau ( E ) normalised to β-actin and expressed as fold change relative to Wt. ( F ) Protein blots of kinases involved in Tau phosphorylation, GSK3, RhoA and Cdk5-activators, p25 and p35 proteins in the forebrain of Wt, p75KO, pR5, and pR75KO mice; of cleaved caspase-3; and of post-synaptic protein, PSD-95 and pre-synaptic proteins, SNAP25 and VAMP2, GFAP, TH ChAT. Protein band intensity quantification of inactive GSK3: GSK3β pS9 normalised with total GSK3β ( G ), active RhoA-GTP normalised with total RhoA ( H ), and Cdk5 activators, p25/p35 ratio ( I ). All band intensities showing ( G – I ) are expressed as fold change relative to Wt. Protein band intensity quantification of cleaved caspase-3 levels ( J ), PSD-95 ( K ), SNAP25 ( L ), VAMP2 ( M ), TH ( N ), GFAP ( O ), ChAT ( P ) normalized with their respective β-actin and expressed as fold change relative to Wt. Data are represented as the mean ± SEM, n=3. Statistical comparisons were performed using one-way ANOVA and Tukey’s test. For human pTau, two-tailed unpaired t-test was used to compare pR5 and pR75KO mice Statistical significance: *P<0.05, **P<0.01 , ***P<0.001, ****P<0.0001 .

Article Snippet: MN1020, CiteAb, UK); sheep anti-human Tau (Antibody Technology Australia, Australia); cleaved caspase-3, rabbit anti-GSK3α/β (Ser21/9), rabbit anti-GSK3β pS9, rabbit anti-p25/35, anti-phosphorylated PKA T197, and anti-total PKA C-α (Cell Signalling Technology, QLD, Australia); rabbit affinity purified anti-ChAT was from Dr . John Oliver (Centre for Neuroscience, Department of Human Physiology, Flinders University); mouse anti-tyrosine hydroxylase (TH) (Sigma-Aldrich, St Louis, MO, US) anti-neuronal nuclei antigen (anti-NeuN) (Merck Millipore, VIC, Australia); rabbit anti-glial fibrillary acidic protein (anti-GFAP) (DAKO, Denmark); rabbit anti-vesicle-associated membrane protein 2 (VAMP2) and rabbit anti-synaptosomal-associated protein 25 (SNAP25) (OSS00035W, Osenses, Australia); anti-postsynaptic density protein 95 (PSD-95) (Sigma-Aldrich, St Louis, MO, US);and mouse monoclonal anti-RhoA-GTP and rabbit anti-total RhoA (New-East Biosciences, Malvern, Pennsylvania, US).

Techniques: Knock-Out, Two Tailed Test