par3 Search Results


par 3  (ATCC)
93
ATCC par 3
Par 3, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par3/pmc02614381-105-9-15?v=ATCC
Average 93 stars, based on 1 article reviews
par 3 - by Bioz Stars, 2026-08
93/100 stars
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pard3α  (Novus Biologicals)
94
Novus Biologicals pard3α
Pard3α, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par3/pm28350047-77-5-8?v=Novus+Biologicals
Average 94 stars, based on 1 article reviews
pard3α - by Bioz Stars, 2026-08
94/100 stars
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par3 sirnas  (Santa Cruz Biotechnology)
92
Santa Cruz Biotechnology par3 sirnas
Expression of protease-activated receptors. After thrombin preconditioning (2 U) for 6 h, umbilical cord-derived mesenchymal stem cells (UCB-MSCs) were lysed and subjected to immunoblotting analysis with the indicated antibodies. ( A ) Immunoblot analysis of the expression of protease-activated receptors (PARs) in UCB-MSCs. After thrombin preconditioning, the levels of PARs in the UCB-MSCs were determined by immunoblotting analysis. ( B ) Bar graph showing quantification of the amounts of each PAR. ( C ) Lysates from UCB-MSCs treated with the indicated <t>siRNAs</t> for 24 h were subjected to immunoblot analysis. Cell lysates were analyzed by immunoblotting, and the protein levels were normalized to GAPDH. An asterisk (*) indicates a significant difference vs. naive EVs.
Par3 Sirnas, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par3/pmc06627943-98-2-7?v=Santa+Cruz+Biotechnology
Average 92 stars, based on 1 article reviews
par3 sirnas - by Bioz Stars, 2026-08
92/100 stars
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rabbit polyclonal anti pard3 par3  (Novus Biologicals)
94
Novus Biologicals rabbit polyclonal anti pard3 par3
KEY RESOURCES TABLE
Rabbit Polyclonal Anti Pard3 Par3, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par3/pmc08604081-23-0-5?v=Novus+Biologicals
Average 94 stars, based on 1 article reviews
rabbit polyclonal anti pard3 par3 - by Bioz Stars, 2026-08
94/100 stars
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polyclonal rabbit anti par3 antibody  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology polyclonal rabbit anti par3 antibody
Figure 1. Identification of ERK2 as a novel <t>Par3-interacting</t> protein. A, Schematic representation of Par3. The numbers indicate the amino acids. CR1, Conserved region 1; PDZ, PSD-95/Dlg/ZO-1; aPKC BR, aPKC binding region. B, Validation of the results of the proteomic analysis. The eluates from affinity column chromatography were analyzed by immuno- blotting using anti-ERK1/2, anti-JNK1, anti-p38 MAPK, anti-GSK-3, and anti-Cdk5 antibodies. C, A coimmunoprecipitation assay was performed using rat brain lysate. Extracts of developing rat brain were incubated with rabbit IgG, anti-Par3, or anti-ERK1/2 antibody. The immunoprecipitates were analyzed by immunoblotting with rabbit anti-Par3 and mouse anti-ERK2, anti-JNK1, anti-p38 MAPK, anti-GSK-3, and anti-Cdk5 antibodies. D, Super-resolution microscopy images of the cell body and the growth cone showing the colocalization ofPar3andERK2.Hippocampalneuronswerefixedat3DIVandthenimmunostainedwithspecificantibodiesagainstanti-ERK2(green)andanti-Par3(red).Theperipheralregionsofthe growth cones were visualized by staining F-actin with Alexa-647-conjugated phalloidin (blue). Arrowheads in the enlarged images indicate the colocalization of the proteins. Scale bars, 5 m and 0.5 m for the enlarged images. E, GST-ERK2 was incubated with amylose-resin coated with MBP, MBP-Par3-1N, -2N, -3N, or -4N. The bound proteins were subjected to immunoblotting with anti-ERK2 antibody (top). The total amounts of MBP, MBP-Par3-1N, -2N, -3N, and -4N are shown with Coomassie Brilliant Blue (CBB) staining (bottom). Asterisks indicate intact MBP-fusion proteins. F, The putative ERK docking site KIM ((V/L)-X2-(R/K)-(R/K)-X3– 6-L).
Polyclonal Rabbit Anti Par3 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par3/10__1523_slash_jneurosci__4210___12__2013-58-7-31?v=Santa+Cruz+Biotechnology
Average 93 stars, based on 1 article reviews
polyclonal rabbit anti par3 antibody - by Bioz Stars, 2026-08
93/100 stars
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par3 proteintech 11085 1 ap rabbit  (Proteintech)
93
Proteintech par3 proteintech 11085 1 ap rabbit
Figure 1. Identification of ERK2 as a novel <t>Par3-interacting</t> protein. A, Schematic representation of Par3. The numbers indicate the amino acids. CR1, Conserved region 1; PDZ, PSD-95/Dlg/ZO-1; aPKC BR, aPKC binding region. B, Validation of the results of the proteomic analysis. The eluates from affinity column chromatography were analyzed by immuno- blotting using anti-ERK1/2, anti-JNK1, anti-p38 MAPK, anti-GSK-3, and anti-Cdk5 antibodies. C, A coimmunoprecipitation assay was performed using rat brain lysate. Extracts of developing rat brain were incubated with rabbit IgG, anti-Par3, or anti-ERK1/2 antibody. The immunoprecipitates were analyzed by immunoblotting with rabbit anti-Par3 and mouse anti-ERK2, anti-JNK1, anti-p38 MAPK, anti-GSK-3, and anti-Cdk5 antibodies. D, Super-resolution microscopy images of the cell body and the growth cone showing the colocalization ofPar3andERK2.Hippocampalneuronswerefixedat3DIVandthenimmunostainedwithspecificantibodiesagainstanti-ERK2(green)andanti-Par3(red).Theperipheralregionsofthe growth cones were visualized by staining F-actin with Alexa-647-conjugated phalloidin (blue). Arrowheads in the enlarged images indicate the colocalization of the proteins. Scale bars, 5 m and 0.5 m for the enlarged images. E, GST-ERK2 was incubated with amylose-resin coated with MBP, MBP-Par3-1N, -2N, -3N, or -4N. The bound proteins were subjected to immunoblotting with anti-ERK2 antibody (top). The total amounts of MBP, MBP-Par3-1N, -2N, -3N, and -4N are shown with Coomassie Brilliant Blue (CBB) staining (bottom). Asterisks indicate intact MBP-fusion proteins. F, The putative ERK docking site KIM ((V/L)-X2-(R/K)-(R/K)-X3– 6-L).
Par3 Proteintech 11085 1 Ap Rabbit, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par3/pmc09751369__Presentation1-24-13-14?v=Proteintech
Average 93 stars, based on 1 article reviews
par3 proteintech 11085 1 ap rabbit - by Bioz Stars, 2026-08
93/100 stars
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par3 agonist peptide  (Biosynth Carbosynth)
90
Biosynth Carbosynth par3 agonist peptide
Figure 1. Identification of ERK2 as a novel <t>Par3-interacting</t> protein. A, Schematic representation of Par3. The numbers indicate the amino acids. CR1, Conserved region 1; PDZ, PSD-95/Dlg/ZO-1; aPKC BR, aPKC binding region. B, Validation of the results of the proteomic analysis. The eluates from affinity column chromatography were analyzed by immuno- blotting using anti-ERK1/2, anti-JNK1, anti-p38 MAPK, anti-GSK-3, and anti-Cdk5 antibodies. C, A coimmunoprecipitation assay was performed using rat brain lysate. Extracts of developing rat brain were incubated with rabbit IgG, anti-Par3, or anti-ERK1/2 antibody. The immunoprecipitates were analyzed by immunoblotting with rabbit anti-Par3 and mouse anti-ERK2, anti-JNK1, anti-p38 MAPK, anti-GSK-3, and anti-Cdk5 antibodies. D, Super-resolution microscopy images of the cell body and the growth cone showing the colocalization ofPar3andERK2.Hippocampalneuronswerefixedat3DIVandthenimmunostainedwithspecificantibodiesagainstanti-ERK2(green)andanti-Par3(red).Theperipheralregionsofthe growth cones were visualized by staining F-actin with Alexa-647-conjugated phalloidin (blue). Arrowheads in the enlarged images indicate the colocalization of the proteins. Scale bars, 5 m and 0.5 m for the enlarged images. E, GST-ERK2 was incubated with amylose-resin coated with MBP, MBP-Par3-1N, -2N, -3N, or -4N. The bound proteins were subjected to immunoblotting with anti-ERK2 antibody (top). The total amounts of MBP, MBP-Par3-1N, -2N, -3N, and -4N are shown with Coomassie Brilliant Blue (CBB) staining (bottom). Asterisks indicate intact MBP-fusion proteins. F, The putative ERK docking site KIM ((V/L)-X2-(R/K)-(R/K)-X3– 6-L).
Par3 Agonist Peptide, supplied by Biosynth Carbosynth, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par3/pm39442624-46-16-26?v=Biosynth+Carbosynth
Average 90 stars, based on 1 article reviews
par3 agonist peptide - by Bioz Stars, 2026-08
90/100 stars
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mouse par3  (OriGene)
90
OriGene mouse par3
Fura 2-loaded wild type (black circle), <t>PAR3</t> −/− (gray circle), and PAR3 +/− (white square) platelets were activated with the indicated concentrations of: ( A ) thrombin, (0.001–100 nM, ( B ) AYPGKF (0–2 mM), ( C ) convulxin (0.01–100 nM), or 20 µM of ADP for 10 min at 37°C in the presence of 2 mM of CaCl 2 . The difference between the maximum increase and the basal intracellular Ca 2+ mobilization was measured. The results are the mean (± SD) of three independent experiments (* p <0.05).
Mouse Par3, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par3/pmc03566007-77-3-8?v=OriGene
Average 90 stars, based on 1 article reviews
mouse par3 - by Bioz Stars, 2026-08
90/100 stars
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par3  (St Johns Laboratory)
93
St Johns Laboratory par3
a) Simplified phylogeny of animals shows that acoel brains are likely intermediate between cnidarian diffuse nets and the centralized brains of typical bilaterians. b) Photograph of juvenile Hofstenia miamia . c) Staining with voltage dye reveals a superficial network of dense neuropil (blue arrow) that extends into a sparser posterior nerve net (green arrow). d) Close-up view of neuropil stained sparsely with tubulin dye (orange) reveals that the neuropil (orange) contains many neurites running in parallel, with cellular clusters (cyan) interspersed between neurite bundles. Sensory neurons (likely clusters of H1 cells; bright orange) are set within many of these patches. e) Cross-section of brain stained with a <t>Par3</t> antibody reveals that the brain has two layers: superficial neuropil, and deeper cell bodies that project outward. f) Staining with an ERK antibody (z-projected segmentation overlaid) shows that brain interneurons can be multipolar, with a central cell body generating multiple neurites. g) Cross-section of brain stained with an antibody against β-catenin reveals another sensory neuron class (possibly H2 ) with two projections that innervate brain neuropil. h) Electron microscopy cross-section shows the fine organization of the brain, confirming the relative configuration of tissue types within the head. The superficial neuropil (previously ‘layer 1’) is visible immediately beneath the skin, while neural cell bodies (previously ‘layer 2’) lie deeper in the tissue, internal to body wall muscle (green). Together, these layers compose the brain. i) Electron microscopy close-up of the brain shows dense neuropil; the box is a 6.7×6.7µm square. j) Segmenting neural projections within the highlighted box in (i) reveals over 400 neurites in a single section of neuropil. k) Segmentation of cellular clusters within neuropil allows quantification of brain structure and its variability. l) Quantifying the numbers of cellular clusters across brains reveals that, although cluster numbers increase with age (i.e. days after hatching) and size (i.e. head width, a good proxy for overall body size ), worms vary widely in how many clusters they possess. Linear regression p<0.0001, n=49. Scale bars: 200µm (c), 50µm (d,e), 20µm (f,g), 10µm (h).
Par3, supplied by St Johns Laboratory, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par3/bio_rxiv__2025__10__03__680388-222-3-4?v=St+Johns+Laboratory
Average 93 stars, based on 1 article reviews
par3 - by Bioz Stars, 2026-08
93/100 stars
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mouse p4a1 anti par 3  (Developmental Studies Hybridoma Bank)
91
Developmental Studies Hybridoma Bank mouse p4a1 anti par 3
a) Simplified phylogeny of animals shows that acoel brains are likely intermediate between cnidarian diffuse nets and the centralized brains of typical bilaterians. b) Photograph of juvenile Hofstenia miamia . c) Staining with voltage dye reveals a superficial network of dense neuropil (blue arrow) that extends into a sparser posterior nerve net (green arrow). d) Close-up view of neuropil stained sparsely with tubulin dye (orange) reveals that the neuropil (orange) contains many neurites running in parallel, with cellular clusters (cyan) interspersed between neurite bundles. Sensory neurons (likely clusters of H1 cells; bright orange) are set within many of these patches. e) Cross-section of brain stained with a <t>Par3</t> antibody reveals that the brain has two layers: superficial neuropil, and deeper cell bodies that project outward. f) Staining with an ERK antibody (z-projected segmentation overlaid) shows that brain interneurons can be multipolar, with a central cell body generating multiple neurites. g) Cross-section of brain stained with an antibody against β-catenin reveals another sensory neuron class (possibly H2 ) with two projections that innervate brain neuropil. h) Electron microscopy cross-section shows the fine organization of the brain, confirming the relative configuration of tissue types within the head. The superficial neuropil (previously ‘layer 1’) is visible immediately beneath the skin, while neural cell bodies (previously ‘layer 2’) lie deeper in the tissue, internal to body wall muscle (green). Together, these layers compose the brain. i) Electron microscopy close-up of the brain shows dense neuropil; the box is a 6.7×6.7µm square. j) Segmenting neural projections within the highlighted box in (i) reveals over 400 neurites in a single section of neuropil. k) Segmentation of cellular clusters within neuropil allows quantification of brain structure and its variability. l) Quantifying the numbers of cellular clusters across brains reveals that, although cluster numbers increase with age (i.e. days after hatching) and size (i.e. head width, a good proxy for overall body size ), worms vary widely in how many clusters they possess. Linear regression p<0.0001, n=49. Scale bars: 200µm (c), 50µm (d,e), 20µm (f,g), 10µm (h).
Mouse P4a1 Anti Par 3, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par3/pm18652816-61-42-52?v=Developmental+Studies+Hybridoma+Bank
Average 91 stars, based on 1 article reviews
mouse p4a1 anti par 3 - by Bioz Stars, 2026-08
91/100 stars
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lentiviral supernatant  (Santa Cruz Biotechnology)
85
Santa Cruz Biotechnology lentiviral supernatant
a) Simplified phylogeny of animals shows that acoel brains are likely intermediate between cnidarian diffuse nets and the centralized brains of typical bilaterians. b) Photograph of juvenile Hofstenia miamia . c) Staining with voltage dye reveals a superficial network of dense neuropil (blue arrow) that extends into a sparser posterior nerve net (green arrow). d) Close-up view of neuropil stained sparsely with tubulin dye (orange) reveals that the neuropil (orange) contains many neurites running in parallel, with cellular clusters (cyan) interspersed between neurite bundles. Sensory neurons (likely clusters of H1 cells; bright orange) are set within many of these patches. e) Cross-section of brain stained with a <t>Par3</t> antibody reveals that the brain has two layers: superficial neuropil, and deeper cell bodies that project outward. f) Staining with an ERK antibody (z-projected segmentation overlaid) shows that brain interneurons can be multipolar, with a central cell body generating multiple neurites. g) Cross-section of brain stained with an antibody against β-catenin reveals another sensory neuron class (possibly H2 ) with two projections that innervate brain neuropil. h) Electron microscopy cross-section shows the fine organization of the brain, confirming the relative configuration of tissue types within the head. The superficial neuropil (previously ‘layer 1’) is visible immediately beneath the skin, while neural cell bodies (previously ‘layer 2’) lie deeper in the tissue, internal to body wall muscle (green). Together, these layers compose the brain. i) Electron microscopy close-up of the brain shows dense neuropil; the box is a 6.7×6.7µm square. j) Segmenting neural projections within the highlighted box in (i) reveals over 400 neurites in a single section of neuropil. k) Segmentation of cellular clusters within neuropil allows quantification of brain structure and its variability. l) Quantifying the numbers of cellular clusters across brains reveals that, although cluster numbers increase with age (i.e. days after hatching) and size (i.e. head width, a good proxy for overall body size ), worms vary widely in how many clusters they possess. Linear regression p<0.0001, n=49. Scale bars: 200µm (c), 50µm (d,e), 20µm (f,g), 10µm (h).
Lentiviral Supernatant, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par3/pmc04947177-209-13-20?v=Santa+Cruz+Biotechnology
Average 85 stars, based on 1 article reviews
lentiviral supernatant - by Bioz Stars, 2026-08
85/100 stars
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gfp par3  (Addgene inc)
92
Addgene inc gfp par3
a) Simplified phylogeny of animals shows that acoel brains are likely intermediate between cnidarian diffuse nets and the centralized brains of typical bilaterians. b) Photograph of juvenile Hofstenia miamia . c) Staining with voltage dye reveals a superficial network of dense neuropil (blue arrow) that extends into a sparser posterior nerve net (green arrow). d) Close-up view of neuropil stained sparsely with tubulin dye (orange) reveals that the neuropil (orange) contains many neurites running in parallel, with cellular clusters (cyan) interspersed between neurite bundles. Sensory neurons (likely clusters of H1 cells; bright orange) are set within many of these patches. e) Cross-section of brain stained with a <t>Par3</t> antibody reveals that the brain has two layers: superficial neuropil, and deeper cell bodies that project outward. f) Staining with an ERK antibody (z-projected segmentation overlaid) shows that brain interneurons can be multipolar, with a central cell body generating multiple neurites. g) Cross-section of brain stained with an antibody against β-catenin reveals another sensory neuron class (possibly H2 ) with two projections that innervate brain neuropil. h) Electron microscopy cross-section shows the fine organization of the brain, confirming the relative configuration of tissue types within the head. The superficial neuropil (previously ‘layer 1’) is visible immediately beneath the skin, while neural cell bodies (previously ‘layer 2’) lie deeper in the tissue, internal to body wall muscle (green). Together, these layers compose the brain. i) Electron microscopy close-up of the brain shows dense neuropil; the box is a 6.7×6.7µm square. j) Segmenting neural projections within the highlighted box in (i) reveals over 400 neurites in a single section of neuropil. k) Segmentation of cellular clusters within neuropil allows quantification of brain structure and its variability. l) Quantifying the numbers of cellular clusters across brains reveals that, although cluster numbers increase with age (i.e. days after hatching) and size (i.e. head width, a good proxy for overall body size ), worms vary widely in how many clusters they possess. Linear regression p<0.0001, n=49. Scale bars: 200µm (c), 50µm (d,e), 20µm (f,g), 10µm (h).
Gfp Par3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/par3/pmc11244941__NIHPP2024__06__30__601440v1___supplement___1-7-66-93?v=Addgene+inc
Average 92 stars, based on 1 article reviews
gfp par3 - by Bioz Stars, 2026-08
92/100 stars
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Image Search Results


Expression of protease-activated receptors. After thrombin preconditioning (2 U) for 6 h, umbilical cord-derived mesenchymal stem cells (UCB-MSCs) were lysed and subjected to immunoblotting analysis with the indicated antibodies. ( A ) Immunoblot analysis of the expression of protease-activated receptors (PARs) in UCB-MSCs. After thrombin preconditioning, the levels of PARs in the UCB-MSCs were determined by immunoblotting analysis. ( B ) Bar graph showing quantification of the amounts of each PAR. ( C ) Lysates from UCB-MSCs treated with the indicated siRNAs for 24 h were subjected to immunoblot analysis. Cell lysates were analyzed by immunoblotting, and the protein levels were normalized to GAPDH. An asterisk (*) indicates a significant difference vs. naive EVs.

Journal: International Journal of Molecular Sciences

Article Title: Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways

doi: 10.3390/ijms20122899

Figure Lengend Snippet: Expression of protease-activated receptors. After thrombin preconditioning (2 U) for 6 h, umbilical cord-derived mesenchymal stem cells (UCB-MSCs) were lysed and subjected to immunoblotting analysis with the indicated antibodies. ( A ) Immunoblot analysis of the expression of protease-activated receptors (PARs) in UCB-MSCs. After thrombin preconditioning, the levels of PARs in the UCB-MSCs were determined by immunoblotting analysis. ( B ) Bar graph showing quantification of the amounts of each PAR. ( C ) Lysates from UCB-MSCs treated with the indicated siRNAs for 24 h were subjected to immunoblot analysis. Cell lysates were analyzed by immunoblotting, and the protein levels were normalized to GAPDH. An asterisk (*) indicates a significant difference vs. naive EVs.

Article Snippet: Control and PAR3 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Derivative Assay, Western Blot

Protease-activated receptors, PAR1 and PAR3, are involved in extracellular vesicle production and phosphorylation of ERK and AKT in umbilical cord-derived mesenchymal stem cells after thrombin preconditioning. Thrombin-preconditioned umbilical cord-derived mesenchymal stem cells (UCB-MSCs) were lysed for immunoblotting analysis with the indicated antibodies. ( A ) After thrombin treatment and inhibition, changes in the protein levels of endosome markers in the UCB-MSCs were assessed by immunoblotting. ( B ) Bar graph showing quantification of Rab-5 and EEA1 levels. ( C ) After thrombin treatment and inhibition, changes in the protein levels of phosphorylated (p)ERK1/2, p AKT, ERK1/2, and AKT in the UCB-MSCs were assessed by immunoblotting. ( D ) Bar graph showing quantification of p ERK1/2 p AKT, ERK1/2, and AKT. ( E ) After thrombin treatment and inhibition, changes in the levels of endosome markers in the UCB-MSCs were determined by immunoblotting. ( F ) Bar graph showing quantification of Rab-5 and EEA1 levels. ( G ) After thrombin treatment, changes in the expression of p ERK1/2 and p AKT in the UCB-MSCs were determined by immunoblotting. ( H ) Bar graph showing quantification of p ERK1/2 and p AKT levels. ( I ) Bar graph showing quantification of the ratio of p ERK/ERK and p AKT/AKT. Data are presented as mean ± SD. An asterisk (*) indicates a significant difference vs. naive EVs, a number sign (#) indicates a significant difference vs. thrombin-treated UCB-MSC and, a dollar sign ($) indicates a significant difference vs. thrombin + PAR3 siRNA UCB-MSCs ( p < 0.05, two-sample t -test; n = 5 per analysis).

Journal: International Journal of Molecular Sciences

Article Title: Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways

doi: 10.3390/ijms20122899

Figure Lengend Snippet: Protease-activated receptors, PAR1 and PAR3, are involved in extracellular vesicle production and phosphorylation of ERK and AKT in umbilical cord-derived mesenchymal stem cells after thrombin preconditioning. Thrombin-preconditioned umbilical cord-derived mesenchymal stem cells (UCB-MSCs) were lysed for immunoblotting analysis with the indicated antibodies. ( A ) After thrombin treatment and inhibition, changes in the protein levels of endosome markers in the UCB-MSCs were assessed by immunoblotting. ( B ) Bar graph showing quantification of Rab-5 and EEA1 levels. ( C ) After thrombin treatment and inhibition, changes in the protein levels of phosphorylated (p)ERK1/2, p AKT, ERK1/2, and AKT in the UCB-MSCs were assessed by immunoblotting. ( D ) Bar graph showing quantification of p ERK1/2 p AKT, ERK1/2, and AKT. ( E ) After thrombin treatment and inhibition, changes in the levels of endosome markers in the UCB-MSCs were determined by immunoblotting. ( F ) Bar graph showing quantification of Rab-5 and EEA1 levels. ( G ) After thrombin treatment, changes in the expression of p ERK1/2 and p AKT in the UCB-MSCs were determined by immunoblotting. ( H ) Bar graph showing quantification of p ERK1/2 and p AKT levels. ( I ) Bar graph showing quantification of the ratio of p ERK/ERK and p AKT/AKT. Data are presented as mean ± SD. An asterisk (*) indicates a significant difference vs. naive EVs, a number sign (#) indicates a significant difference vs. thrombin-treated UCB-MSC and, a dollar sign ($) indicates a significant difference vs. thrombin + PAR3 siRNA UCB-MSCs ( p < 0.05, two-sample t -test; n = 5 per analysis).

Article Snippet: Control and PAR3 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Derivative Assay, Western Blot, Inhibition, Expressing

Protease-activated receptors, PAR1 and PAR3, are involved in endosome production by umbilical cord-derived mesenchymal stem cells and protein cargo contents in extracellular vesicles after thrombin preconditioning. ( A ) After thrombin preconditioning of umbilical cord-derived mesenchymal stem cells (UCB-MSCs), the number of endosomes was determined by labeling early endosomes with green fluorescent protein(GFP-green) and the nuclei with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI-blue). ( B ) Bar graph showing the quantified intensities of the endosome-GFP signal. ( C ) After treatment with thrombin and thrombin inhibitor, the number of extracellular vesicles (EVs) was determined using a NanoSightNS300. ( D ) The size and number distribution of EVs as measured and analyzed by Nanoparticle Tracking Analysis software. ( E ) After treatment with thrombin and thrombin inhibitor, the levels of VEGF, angiogenin, angiopoietin, and HGF in the EVs were measured by multiplex ELISA. Following treatment with thrombin and thrombin inhibition, EVs were isolated from the conditioned media of UCB-MSC cultures, and VEGF, angiogenin, angiopoietin, and HGF protein levels were assessed. An asterisk (*) indicates a significant difference vs. naive EVs, a number sign (#) indicates a significant difference vs. thrombin-treated UCB-MSC and a dollar sign ($) indicates a significant difference vs. thrombin + PAR3 siRNA UCB-MSC ( p < 0.05, two-sample t -test; n = 6 per analysis).

Journal: International Journal of Molecular Sciences

Article Title: Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways

doi: 10.3390/ijms20122899

Figure Lengend Snippet: Protease-activated receptors, PAR1 and PAR3, are involved in endosome production by umbilical cord-derived mesenchymal stem cells and protein cargo contents in extracellular vesicles after thrombin preconditioning. ( A ) After thrombin preconditioning of umbilical cord-derived mesenchymal stem cells (UCB-MSCs), the number of endosomes was determined by labeling early endosomes with green fluorescent protein(GFP-green) and the nuclei with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI-blue). ( B ) Bar graph showing the quantified intensities of the endosome-GFP signal. ( C ) After treatment with thrombin and thrombin inhibitor, the number of extracellular vesicles (EVs) was determined using a NanoSightNS300. ( D ) The size and number distribution of EVs as measured and analyzed by Nanoparticle Tracking Analysis software. ( E ) After treatment with thrombin and thrombin inhibitor, the levels of VEGF, angiogenin, angiopoietin, and HGF in the EVs were measured by multiplex ELISA. Following treatment with thrombin and thrombin inhibition, EVs were isolated from the conditioned media of UCB-MSC cultures, and VEGF, angiogenin, angiopoietin, and HGF protein levels were assessed. An asterisk (*) indicates a significant difference vs. naive EVs, a number sign (#) indicates a significant difference vs. thrombin-treated UCB-MSC and a dollar sign ($) indicates a significant difference vs. thrombin + PAR3 siRNA UCB-MSC ( p < 0.05, two-sample t -test; n = 6 per analysis).

Article Snippet: Control and PAR3 siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Derivative Assay, Labeling, Software, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Isolation

KEY RESOURCES TABLE

Journal: Cell reports

Article Title: Cdc42 activity in Sertoli cells is essential for maintenance of spermatogenesis

doi: 10.1016/j.celrep.2021.109885

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-PARD3 (Par3) , Novus , Cat#NBP1-88861; RRID: AB_11056253.

Techniques: Recombinant, Microarray, Real-time Polymerase Chain Reaction, Software

Figure 1. Identification of ERK2 as a novel Par3-interacting protein. A, Schematic representation of Par3. The numbers indicate the amino acids. CR1, Conserved region 1; PDZ, PSD-95/Dlg/ZO-1; aPKC BR, aPKC binding region. B, Validation of the results of the proteomic analysis. The eluates from affinity column chromatography were analyzed by immuno- blotting using anti-ERK1/2, anti-JNK1, anti-p38 MAPK, anti-GSK-3, and anti-Cdk5 antibodies. C, A coimmunoprecipitation assay was performed using rat brain lysate. Extracts of developing rat brain were incubated with rabbit IgG, anti-Par3, or anti-ERK1/2 antibody. The immunoprecipitates were analyzed by immunoblotting with rabbit anti-Par3 and mouse anti-ERK2, anti-JNK1, anti-p38 MAPK, anti-GSK-3, and anti-Cdk5 antibodies. D, Super-resolution microscopy images of the cell body and the growth cone showing the colocalization ofPar3andERK2.Hippocampalneuronswerefixedat3DIVandthenimmunostainedwithspecificantibodiesagainstanti-ERK2(green)andanti-Par3(red).Theperipheralregionsofthe growth cones were visualized by staining F-actin with Alexa-647-conjugated phalloidin (blue). Arrowheads in the enlarged images indicate the colocalization of the proteins. Scale bars, 5 m and 0.5 m for the enlarged images. E, GST-ERK2 was incubated with amylose-resin coated with MBP, MBP-Par3-1N, -2N, -3N, or -4N. The bound proteins were subjected to immunoblotting with anti-ERK2 antibody (top). The total amounts of MBP, MBP-Par3-1N, -2N, -3N, and -4N are shown with Coomassie Brilliant Blue (CBB) staining (bottom). Asterisks indicate intact MBP-fusion proteins. F, The putative ERK docking site KIM ((V/L)-X2-(R/K)-(R/K)-X3– 6-L).

Journal: Journal of Neuroscience

Article Title: ERK2-Mediated Phosphorylation of Par3 Regulates Neuronal Polarization

doi: 10.1523/jneurosci.4210-12.2013

Figure Lengend Snippet: Figure 1. Identification of ERK2 as a novel Par3-interacting protein. A, Schematic representation of Par3. The numbers indicate the amino acids. CR1, Conserved region 1; PDZ, PSD-95/Dlg/ZO-1; aPKC BR, aPKC binding region. B, Validation of the results of the proteomic analysis. The eluates from affinity column chromatography were analyzed by immuno- blotting using anti-ERK1/2, anti-JNK1, anti-p38 MAPK, anti-GSK-3, and anti-Cdk5 antibodies. C, A coimmunoprecipitation assay was performed using rat brain lysate. Extracts of developing rat brain were incubated with rabbit IgG, anti-Par3, or anti-ERK1/2 antibody. The immunoprecipitates were analyzed by immunoblotting with rabbit anti-Par3 and mouse anti-ERK2, anti-JNK1, anti-p38 MAPK, anti-GSK-3, and anti-Cdk5 antibodies. D, Super-resolution microscopy images of the cell body and the growth cone showing the colocalization ofPar3andERK2.Hippocampalneuronswerefixedat3DIVandthenimmunostainedwithspecificantibodiesagainstanti-ERK2(green)andanti-Par3(red).Theperipheralregionsofthe growth cones were visualized by staining F-actin with Alexa-647-conjugated phalloidin (blue). Arrowheads in the enlarged images indicate the colocalization of the proteins. Scale bars, 5 m and 0.5 m for the enlarged images. E, GST-ERK2 was incubated with amylose-resin coated with MBP, MBP-Par3-1N, -2N, -3N, or -4N. The bound proteins were subjected to immunoblotting with anti-ERK2 antibody (top). The total amounts of MBP, MBP-Par3-1N, -2N, -3N, and -4N are shown with Coomassie Brilliant Blue (CBB) staining (bottom). Asterisks indicate intact MBP-fusion proteins. F, The putative ERK docking site KIM ((V/L)-X2-(R/K)-(R/K)-X3– 6-L).

Article Snippet: The following antibodies and materials were used: polyclonal rabbit anti-Par3 antibody raised against Par3-4N/3S-GST (Nishimura et al., 2004); polyclonal rabbit anti-Par3 and monoclonal mouse anti-Cdk5 antibodies (Millipore); polyclonal rabbit anti-c-Myc antibody (Santa Cruz Biotechnology); polyclonal rabbit anti-phospho-ERK1/2 (T202/Y204), polyclonal rabbit anti-ERK1/2, monoclonal rabbit anti-phospho-MAPK/CDK substrates (PXSP or SPXR/K) (34B2), monoclonal mouse anti-JNK1 (C26), and monoclonal mouse anti-p38 MAPK antibodies (Cell Signaling Technology); monoclonal mouse anti-ERK2, monoclonal mouse anti-KIF3A, and monoclonal mouse anti-GSK-3 antibodies (BD Biosciences); monoclonal mouse anti-KIF3A (K2.4) antibody (Abcam); monoclonal mouse anti-Myc (9E10), monoclonal mouse anti- -tubulin (DM1A), monoclonal mouse anti-GST, and monoclonal mouse anti-Flag antibodies (Sigma); monoclonal mouse anti-Tau-1 (Millipore Bioscience Research Reagents); monoclonal mouse anti-class III -tubulin and polyclonal rabbit anti-class III -tubulin antibodies (Tuj1, Covance); polyclonal rabbit anti-GFP and polyclonal rabbit anti-HA antibodies (MBL); and monoclonal mouse anti-GFP antibody (Roche Diagnostics).

Techniques: Binding Assay, Biomarker Discovery, Affinity Column, Chromatography, Co-Immunoprecipitation Assay, Incubation, Western Blot, Super-Resolution Microscopy, Staining

Figure2. IdentificationofthephosphorylationsiteofPar3byERK2.A,ThedirectphosphorylationofPar3byERK2.PurifiedMBPorMBP-Par3deletionmutantswereincubatedwithrecombinant ERK2inthepresenceof[- 32P]ATPinvitro.SamplesweresubjectedtoSDS-PAGEandsilverstaining(bottom)followedbyautoradiography(top).AsterisksindicateintactMBP-fusionproteins.B, ThephosphorylationofPar3pointmutantsbyERK2.PurifiedMBP,MBP-Par3-4N-WT-S1116A,-T1145A,-S1316A,or-T1328AwasincubatedwithrecombinantERK2inthepresenceof[- 32P]ATP in vitro. Samples were subjected to SDS-PAGE and silver staining (bottom) followed by autoradiography (top). Asterisks indicate intact MBP-fusion proteins. C, The alignment of ERK2 phosphory- lation sites in Par3 homologs (rat, mouse, human). D, The phosphorylation of Par3-WT or -S1116A in COS7 cells. COS7 cells were transfected with EGFP-Par3-WT or -S1116A and cultured for 24 h. The cells were serum-starved for 24 h and then treated with or without 1 M OA for 2 h. Cell lysates were incubated with anti-GFP antibody, and the immunoprecipitates were analyzed by immunoblotting with anti-phospho-MAPK/CDK substrates and anti-GFP antibodies.

Journal: Journal of Neuroscience

Article Title: ERK2-Mediated Phosphorylation of Par3 Regulates Neuronal Polarization

doi: 10.1523/jneurosci.4210-12.2013

Figure Lengend Snippet: Figure2. IdentificationofthephosphorylationsiteofPar3byERK2.A,ThedirectphosphorylationofPar3byERK2.PurifiedMBPorMBP-Par3deletionmutantswereincubatedwithrecombinant ERK2inthepresenceof[- 32P]ATPinvitro.SamplesweresubjectedtoSDS-PAGEandsilverstaining(bottom)followedbyautoradiography(top).AsterisksindicateintactMBP-fusionproteins.B, ThephosphorylationofPar3pointmutantsbyERK2.PurifiedMBP,MBP-Par3-4N-WT-S1116A,-T1145A,-S1316A,or-T1328AwasincubatedwithrecombinantERK2inthepresenceof[- 32P]ATP in vitro. Samples were subjected to SDS-PAGE and silver staining (bottom) followed by autoradiography (top). Asterisks indicate intact MBP-fusion proteins. C, The alignment of ERK2 phosphory- lation sites in Par3 homologs (rat, mouse, human). D, The phosphorylation of Par3-WT or -S1116A in COS7 cells. COS7 cells were transfected with EGFP-Par3-WT or -S1116A and cultured for 24 h. The cells were serum-starved for 24 h and then treated with or without 1 M OA for 2 h. Cell lysates were incubated with anti-GFP antibody, and the immunoprecipitates were analyzed by immunoblotting with anti-phospho-MAPK/CDK substrates and anti-GFP antibodies.

Article Snippet: The following antibodies and materials were used: polyclonal rabbit anti-Par3 antibody raised against Par3-4N/3S-GST (Nishimura et al., 2004); polyclonal rabbit anti-Par3 and monoclonal mouse anti-Cdk5 antibodies (Millipore); polyclonal rabbit anti-c-Myc antibody (Santa Cruz Biotechnology); polyclonal rabbit anti-phospho-ERK1/2 (T202/Y204), polyclonal rabbit anti-ERK1/2, monoclonal rabbit anti-phospho-MAPK/CDK substrates (PXSP or SPXR/K) (34B2), monoclonal mouse anti-JNK1 (C26), and monoclonal mouse anti-p38 MAPK antibodies (Cell Signaling Technology); monoclonal mouse anti-ERK2, monoclonal mouse anti-KIF3A, and monoclonal mouse anti-GSK-3 antibodies (BD Biosciences); monoclonal mouse anti-KIF3A (K2.4) antibody (Abcam); monoclonal mouse anti-Myc (9E10), monoclonal mouse anti- -tubulin (DM1A), monoclonal mouse anti-GST, and monoclonal mouse anti-Flag antibodies (Sigma); monoclonal mouse anti-Tau-1 (Millipore Bioscience Research Reagents); monoclonal mouse anti-class III -tubulin and polyclonal rabbit anti-class III -tubulin antibodies (Tuj1, Covance); polyclonal rabbit anti-GFP and polyclonal rabbit anti-HA antibodies (MBL); and monoclonal mouse anti-GFP antibody (Roche Diagnostics).

Techniques: In Vitro, SDS Page, Silver Staining, Autoradiography, Phospho-proteomics, Transfection, Cell Culture, Incubation, Western Blot

Figure 3. Phosphorylation of Par3 by ERK2 in vivo. A, Specificity of the phosphospecific anti-phospho-Par3 (pS1116) antibody. MBP-Par3-4N-WT (1 pmol) containing the indicated amounts of Par3-4N-WTor-S1116AphosphorylatedbyERK2wassubjectedtoSDS-PAGE.Immunoblotanalyseswithanti-pS1116(top) and anti-MBP antibodies (bottom) were (Figure legend continues.)

Journal: Journal of Neuroscience

Article Title: ERK2-Mediated Phosphorylation of Par3 Regulates Neuronal Polarization

doi: 10.1523/jneurosci.4210-12.2013

Figure Lengend Snippet: Figure 3. Phosphorylation of Par3 by ERK2 in vivo. A, Specificity of the phosphospecific anti-phospho-Par3 (pS1116) antibody. MBP-Par3-4N-WT (1 pmol) containing the indicated amounts of Par3-4N-WTor-S1116AphosphorylatedbyERK2wassubjectedtoSDS-PAGE.Immunoblotanalyseswithanti-pS1116(top) and anti-MBP antibodies (bottom) were (Figure legend continues.)

Article Snippet: The following antibodies and materials were used: polyclonal rabbit anti-Par3 antibody raised against Par3-4N/3S-GST (Nishimura et al., 2004); polyclonal rabbit anti-Par3 and monoclonal mouse anti-Cdk5 antibodies (Millipore); polyclonal rabbit anti-c-Myc antibody (Santa Cruz Biotechnology); polyclonal rabbit anti-phospho-ERK1/2 (T202/Y204), polyclonal rabbit anti-ERK1/2, monoclonal rabbit anti-phospho-MAPK/CDK substrates (PXSP or SPXR/K) (34B2), monoclonal mouse anti-JNK1 (C26), and monoclonal mouse anti-p38 MAPK antibodies (Cell Signaling Technology); monoclonal mouse anti-ERK2, monoclonal mouse anti-KIF3A, and monoclonal mouse anti-GSK-3 antibodies (BD Biosciences); monoclonal mouse anti-KIF3A (K2.4) antibody (Abcam); monoclonal mouse anti-Myc (9E10), monoclonal mouse anti- -tubulin (DM1A), monoclonal mouse anti-GST, and monoclonal mouse anti-Flag antibodies (Sigma); monoclonal mouse anti-Tau-1 (Millipore Bioscience Research Reagents); monoclonal mouse anti-class III -tubulin and polyclonal rabbit anti-class III -tubulin antibodies (Tuj1, Covance); polyclonal rabbit anti-GFP and polyclonal rabbit anti-HA antibodies (MBL); and monoclonal mouse anti-GFP antibody (Roche Diagnostics).

Techniques: Phospho-proteomics, In Vivo

Figure 4. The effect of the phosphorylation of Par3 on the interaction of Par3 with KIF3A. A, Schematic representation of KIF3A. The numbers show the amino acids. B, The effect of ERK2 phosphorylationontheinteractionofPar3withKIF3Ainvitro.PurifiedMBP,MBP-Par3-4N-WT,or-S1116AwasincubatedwithorwithoutrecombinantERK2inthepresenceorabsenceofATP.The reactantswereincubatedwithglutathionebeadscoatedwithGSTorGST-KIF3A-C2.Theboundproteinsweresubjectedtoimmunoblottingwithanti-MBPantibody.ThetotalamountsofGSTand GST-KIF3A-C2 are shown with Coomassie Brilliant Blue staining.C, The interaction of KIF3A with Par3-WT, -1116A, -S1116D, or -4N/2. COS7 cells were transfected with the indicated constructs, serum-starved for 24 h, and then stimulated with or without 1 M OA for 2 h. Extracts of COS7 cells were incubated with anti-GFP antibody. The bound proteins and coimmunoprecipitates were analyzedbyimmunoblottingwithanti-GFPandanti-Mycantibodies.Theproteinexpressionlevelsincelllysatesareshownonthetop.D,TheeffectofthephosphorylationofPar3ontheinteraction ofPar3withKIF3Ainvivo.MousebrainsliceswereculturedandtreatedwithorwithoutNT-3(100ng/ml)or1MOAfor2h.Extractsofmousebrainsliceswereincubatedwithanti-Par3antibody. The immunoprecipitates were analyzed by immunoblotting with anti-Par3, anti-KIF3A, anti-ERK1/2, and anti-phospho-ERK1/2 antibodies. E, The interaction of Par6 and aPKC with Par3-WT or -S1116D. COS7 cell lysates expressing the indicated proteins were incubated with anti-GFP antibody. The bound proteins and coimmunoprecipitates were analyzed by immunoblotting with anti-Myc,anti-Flag,andanti-GFPantibodies.Proteinexpressionlevelsinthecelllysatesareshownontheleft.F,TheinteractionofLIMK2withPar3-WTor-S1116D.COS7celllysatesexpressingthe indicated proteins were incubated with anti-GFP antibody. The bound proteins and coimmunoprecipitates were analyzed by immunoblotting with anti-HA and anti-GFP antibodies. Protein expression levels in the cell lysates are shown on the left.

Journal: Journal of Neuroscience

Article Title: ERK2-Mediated Phosphorylation of Par3 Regulates Neuronal Polarization

doi: 10.1523/jneurosci.4210-12.2013

Figure Lengend Snippet: Figure 4. The effect of the phosphorylation of Par3 on the interaction of Par3 with KIF3A. A, Schematic representation of KIF3A. The numbers show the amino acids. B, The effect of ERK2 phosphorylationontheinteractionofPar3withKIF3Ainvitro.PurifiedMBP,MBP-Par3-4N-WT,or-S1116AwasincubatedwithorwithoutrecombinantERK2inthepresenceorabsenceofATP.The reactantswereincubatedwithglutathionebeadscoatedwithGSTorGST-KIF3A-C2.Theboundproteinsweresubjectedtoimmunoblottingwithanti-MBPantibody.ThetotalamountsofGSTand GST-KIF3A-C2 are shown with Coomassie Brilliant Blue staining.C, The interaction of KIF3A with Par3-WT, -1116A, -S1116D, or -4N/2. COS7 cells were transfected with the indicated constructs, serum-starved for 24 h, and then stimulated with or without 1 M OA for 2 h. Extracts of COS7 cells were incubated with anti-GFP antibody. The bound proteins and coimmunoprecipitates were analyzedbyimmunoblottingwithanti-GFPandanti-Mycantibodies.Theproteinexpressionlevelsincelllysatesareshownonthetop.D,TheeffectofthephosphorylationofPar3ontheinteraction ofPar3withKIF3Ainvivo.MousebrainsliceswereculturedandtreatedwithorwithoutNT-3(100ng/ml)or1MOAfor2h.Extractsofmousebrainsliceswereincubatedwithanti-Par3antibody. The immunoprecipitates were analyzed by immunoblotting with anti-Par3, anti-KIF3A, anti-ERK1/2, and anti-phospho-ERK1/2 antibodies. E, The interaction of Par6 and aPKC with Par3-WT or -S1116D. COS7 cell lysates expressing the indicated proteins were incubated with anti-GFP antibody. The bound proteins and coimmunoprecipitates were analyzed by immunoblotting with anti-Myc,anti-Flag,andanti-GFPantibodies.Proteinexpressionlevelsinthecelllysatesareshownontheleft.F,TheinteractionofLIMK2withPar3-WTor-S1116D.COS7celllysatesexpressingthe indicated proteins were incubated with anti-GFP antibody. The bound proteins and coimmunoprecipitates were analyzed by immunoblotting with anti-HA and anti-GFP antibodies. Protein expression levels in the cell lysates are shown on the left.

Article Snippet: The following antibodies and materials were used: polyclonal rabbit anti-Par3 antibody raised against Par3-4N/3S-GST (Nishimura et al., 2004); polyclonal rabbit anti-Par3 and monoclonal mouse anti-Cdk5 antibodies (Millipore); polyclonal rabbit anti-c-Myc antibody (Santa Cruz Biotechnology); polyclonal rabbit anti-phospho-ERK1/2 (T202/Y204), polyclonal rabbit anti-ERK1/2, monoclonal rabbit anti-phospho-MAPK/CDK substrates (PXSP or SPXR/K) (34B2), monoclonal mouse anti-JNK1 (C26), and monoclonal mouse anti-p38 MAPK antibodies (Cell Signaling Technology); monoclonal mouse anti-ERK2, monoclonal mouse anti-KIF3A, and monoclonal mouse anti-GSK-3 antibodies (BD Biosciences); monoclonal mouse anti-KIF3A (K2.4) antibody (Abcam); monoclonal mouse anti-Myc (9E10), monoclonal mouse anti- -tubulin (DM1A), monoclonal mouse anti-GST, and monoclonal mouse anti-Flag antibodies (Sigma); monoclonal mouse anti-Tau-1 (Millipore Bioscience Research Reagents); monoclonal mouse anti-class III -tubulin and polyclonal rabbit anti-class III -tubulin antibodies (Tuj1, Covance); polyclonal rabbit anti-GFP and polyclonal rabbit anti-HA antibodies (MBL); and monoclonal mouse anti-GFP antibody (Roche Diagnostics).

Techniques: Phospho-proteomics, Staining, Transfection, Construct, Incubation, Western Blot, Expressing

Figure5. TheeffectofmutationsofPar3atphosphorylationsitesonaxonaltransportofPar3.A,Super-resolutionmicroscopyimagesoftheaxonalshaftandthegrowthcone(GC)showingthe colocalizationofPar3andKIF3Aonmicrotubules.Hippocampalneuronswerefixedat3DIVandthenimmunostainedwithspecificantibodiesagainstrabbitanti-Par3(red,topandbottom),rabbit or mouse anti-class III -tubulin (green, top and middle), and mouse anti-KIF3A (red, middle; green, bottom). Arrowheads in the enlarged images indicate the colocalization of the proteins. The peripheral regions of the growth cones were visualized by staining F-actin with Alexa-647-conjugated phalloidin (blue). Scale bars: left, 5 m; right, 0.5 m. B, The interaction of Par3-WT or -S1116DwithKIF3Aonmicrotubules.COS7cellsweretransfectedwiththeindicatedconstructs,andthelysatesweresubjectedtothemicrotubulecosedimentationassay.(Figurelegendcontinues.)

Journal: Journal of Neuroscience

Article Title: ERK2-Mediated Phosphorylation of Par3 Regulates Neuronal Polarization

doi: 10.1523/jneurosci.4210-12.2013

Figure Lengend Snippet: Figure5. TheeffectofmutationsofPar3atphosphorylationsitesonaxonaltransportofPar3.A,Super-resolutionmicroscopyimagesoftheaxonalshaftandthegrowthcone(GC)showingthe colocalizationofPar3andKIF3Aonmicrotubules.Hippocampalneuronswerefixedat3DIVandthenimmunostainedwithspecificantibodiesagainstrabbitanti-Par3(red,topandbottom),rabbit or mouse anti-class III -tubulin (green, top and middle), and mouse anti-KIF3A (red, middle; green, bottom). Arrowheads in the enlarged images indicate the colocalization of the proteins. The peripheral regions of the growth cones were visualized by staining F-actin with Alexa-647-conjugated phalloidin (blue). Scale bars: left, 5 m; right, 0.5 m. B, The interaction of Par3-WT or -S1116DwithKIF3Aonmicrotubules.COS7cellsweretransfectedwiththeindicatedconstructs,andthelysatesweresubjectedtothemicrotubulecosedimentationassay.(Figurelegendcontinues.)

Article Snippet: The following antibodies and materials were used: polyclonal rabbit anti-Par3 antibody raised against Par3-4N/3S-GST (Nishimura et al., 2004); polyclonal rabbit anti-Par3 and monoclonal mouse anti-Cdk5 antibodies (Millipore); polyclonal rabbit anti-c-Myc antibody (Santa Cruz Biotechnology); polyclonal rabbit anti-phospho-ERK1/2 (T202/Y204), polyclonal rabbit anti-ERK1/2, monoclonal rabbit anti-phospho-MAPK/CDK substrates (PXSP or SPXR/K) (34B2), monoclonal mouse anti-JNK1 (C26), and monoclonal mouse anti-p38 MAPK antibodies (Cell Signaling Technology); monoclonal mouse anti-ERK2, monoclonal mouse anti-KIF3A, and monoclonal mouse anti-GSK-3 antibodies (BD Biosciences); monoclonal mouse anti-KIF3A (K2.4) antibody (Abcam); monoclonal mouse anti-Myc (9E10), monoclonal mouse anti- -tubulin (DM1A), monoclonal mouse anti-GST, and monoclonal mouse anti-Flag antibodies (Sigma); monoclonal mouse anti-Tau-1 (Millipore Bioscience Research Reagents); monoclonal mouse anti-class III -tubulin and polyclonal rabbit anti-class III -tubulin antibodies (Tuj1, Covance); polyclonal rabbit anti-GFP and polyclonal rabbit anti-HA antibodies (MBL); and monoclonal mouse anti-GFP antibody (Roche Diagnostics).

Techniques: Staining

Figure 7. The effect of mutations of Par3 at phosphorylation sites on neuronal polarity. A–C, Hippocampal neurons were cotransfected with siScramble or siPar3 #6 and the indicated RNAi-resistant Myc-tagged Par3 constructs. A, Representative images of neurons at 3 DIV are shown. Scale bars, 20 m. B, The length of the longest neurite. C, The (Figure legend continues.)

Journal: Journal of Neuroscience

Article Title: ERK2-Mediated Phosphorylation of Par3 Regulates Neuronal Polarization

doi: 10.1523/jneurosci.4210-12.2013

Figure Lengend Snippet: Figure 7. The effect of mutations of Par3 at phosphorylation sites on neuronal polarity. A–C, Hippocampal neurons were cotransfected with siScramble or siPar3 #6 and the indicated RNAi-resistant Myc-tagged Par3 constructs. A, Representative images of neurons at 3 DIV are shown. Scale bars, 20 m. B, The length of the longest neurite. C, The (Figure legend continues.)

Article Snippet: The following antibodies and materials were used: polyclonal rabbit anti-Par3 antibody raised against Par3-4N/3S-GST (Nishimura et al., 2004); polyclonal rabbit anti-Par3 and monoclonal mouse anti-Cdk5 antibodies (Millipore); polyclonal rabbit anti-c-Myc antibody (Santa Cruz Biotechnology); polyclonal rabbit anti-phospho-ERK1/2 (T202/Y204), polyclonal rabbit anti-ERK1/2, monoclonal rabbit anti-phospho-MAPK/CDK substrates (PXSP or SPXR/K) (34B2), monoclonal mouse anti-JNK1 (C26), and monoclonal mouse anti-p38 MAPK antibodies (Cell Signaling Technology); monoclonal mouse anti-ERK2, monoclonal mouse anti-KIF3A, and monoclonal mouse anti-GSK-3 antibodies (BD Biosciences); monoclonal mouse anti-KIF3A (K2.4) antibody (Abcam); monoclonal mouse anti-Myc (9E10), monoclonal mouse anti- -tubulin (DM1A), monoclonal mouse anti-GST, and monoclonal mouse anti-Flag antibodies (Sigma); monoclonal mouse anti-Tau-1 (Millipore Bioscience Research Reagents); monoclonal mouse anti-class III -tubulin and polyclonal rabbit anti-class III -tubulin antibodies (Tuj1, Covance); polyclonal rabbit anti-GFP and polyclonal rabbit anti-HA antibodies (MBL); and monoclonal mouse anti-GFP antibody (Roche Diagnostics).

Techniques: Phospho-proteomics, Construct

Figure8. TheeffectofmutationsofPar3atphosphorylationsitesonneuronalpolarityinvivo.A,pSico-mCherry(shControl),pSico-mCherry-shPar3#1,-shPar3#2,-shPar3#3,or-shPar3#4were cotransfectedintoNeuro2acellswithpEF-Cre.After72h,thecellswerelysedandsubjectedtoimmunoblottingwithanti-Par3andanti--tubulinantibodies.B,pEGFP-Par3orRNA-interference- resistantpEGFP-Par3(pEGFP-rrPar3)werecotransfectedintoCOS7cellswithpSico-mCherryorpSico-mCherry-shPar3#3withpEF-Cre.After72h,thecellswerelysed(Figurelegendcontinues.)

Journal: Journal of Neuroscience

Article Title: ERK2-Mediated Phosphorylation of Par3 Regulates Neuronal Polarization

doi: 10.1523/jneurosci.4210-12.2013

Figure Lengend Snippet: Figure8. TheeffectofmutationsofPar3atphosphorylationsitesonneuronalpolarityinvivo.A,pSico-mCherry(shControl),pSico-mCherry-shPar3#1,-shPar3#2,-shPar3#3,or-shPar3#4were cotransfectedintoNeuro2acellswithpEF-Cre.After72h,thecellswerelysedandsubjectedtoimmunoblottingwithanti-Par3andanti--tubulinantibodies.B,pEGFP-Par3orRNA-interference- resistantpEGFP-Par3(pEGFP-rrPar3)werecotransfectedintoCOS7cellswithpSico-mCherryorpSico-mCherry-shPar3#3withpEF-Cre.After72h,thecellswerelysed(Figurelegendcontinues.)

Article Snippet: The following antibodies and materials were used: polyclonal rabbit anti-Par3 antibody raised against Par3-4N/3S-GST (Nishimura et al., 2004); polyclonal rabbit anti-Par3 and monoclonal mouse anti-Cdk5 antibodies (Millipore); polyclonal rabbit anti-c-Myc antibody (Santa Cruz Biotechnology); polyclonal rabbit anti-phospho-ERK1/2 (T202/Y204), polyclonal rabbit anti-ERK1/2, monoclonal rabbit anti-phospho-MAPK/CDK substrates (PXSP or SPXR/K) (34B2), monoclonal mouse anti-JNK1 (C26), and monoclonal mouse anti-p38 MAPK antibodies (Cell Signaling Technology); monoclonal mouse anti-ERK2, monoclonal mouse anti-KIF3A, and monoclonal mouse anti-GSK-3 antibodies (BD Biosciences); monoclonal mouse anti-KIF3A (K2.4) antibody (Abcam); monoclonal mouse anti-Myc (9E10), monoclonal mouse anti- -tubulin (DM1A), monoclonal mouse anti-GST, and monoclonal mouse anti-Flag antibodies (Sigma); monoclonal mouse anti-Tau-1 (Millipore Bioscience Research Reagents); monoclonal mouse anti-class III -tubulin and polyclonal rabbit anti-class III -tubulin antibodies (Tuj1, Covance); polyclonal rabbit anti-GFP and polyclonal rabbit anti-HA antibodies (MBL); and monoclonal mouse anti-GFP antibody (Roche Diagnostics).

Techniques:

Fura 2-loaded wild type (black circle), PAR3 −/− (gray circle), and PAR3 +/− (white square) platelets were activated with the indicated concentrations of: ( A ) thrombin, (0.001–100 nM, ( B ) AYPGKF (0–2 mM), ( C ) convulxin (0.01–100 nM), or 20 µM of ADP for 10 min at 37°C in the presence of 2 mM of CaCl 2 . The difference between the maximum increase and the basal intracellular Ca 2+ mobilization was measured. The results are the mean (± SD) of three independent experiments (* p <0.05).

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Fura 2-loaded wild type (black circle), PAR3 −/− (gray circle), and PAR3 +/− (white square) platelets were activated with the indicated concentrations of: ( A ) thrombin, (0.001–100 nM, ( B ) AYPGKF (0–2 mM), ( C ) convulxin (0.01–100 nM), or 20 µM of ADP for 10 min at 37°C in the presence of 2 mM of CaCl 2 . The difference between the maximum increase and the basal intracellular Ca 2+ mobilization was measured. The results are the mean (± SD) of three independent experiments (* p <0.05).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques:

Flow cytometric analysis of PAR4 expression in wild type (WT) (black line), PAR3 −/− (gray line), and PAR4 −/− (shaded) mice platelets using anti-PAR4-FITC antibodies.

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Flow cytometric analysis of PAR4 expression in wild type (WT) (black line), PAR3 −/− (gray line), and PAR4 −/− (shaded) mice platelets using anti-PAR4-FITC antibodies.

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Expressing

Fura 2-loaded wild type (black) and PAR3 −/− (gray) platelets were incubated at 37°C for 5 min in the absence or the presence of 100 µM 2MeSAMP. After treatment, platelets were activated 100 nM thrombin ( A ,) or 2 mM AYPGKF ( B ) for 10 min at 37°C in the presence of 2 mM of CaCl 2 . The difference between the maximum increase and the basal intracellular Ca 2+ mobilization was measured. The results are the mean (± SD) of three independent experiments (* p <0.05).

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Fura 2-loaded wild type (black) and PAR3 −/− (gray) platelets were incubated at 37°C for 5 min in the absence or the presence of 100 µM 2MeSAMP. After treatment, platelets were activated 100 nM thrombin ( A ,) or 2 mM AYPGKF ( B ) for 10 min at 37°C in the presence of 2 mM of CaCl 2 . The difference between the maximum increase and the basal intracellular Ca 2+ mobilization was measured. The results are the mean (± SD) of three independent experiments (* p <0.05).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Incubation

Fura 2-loaded wild type (black line) and PAR3 −/− (gray line) platelets were resuspended in Ca 2+ -free medium (0.1 mM EGTA was added at the time of experiment). Representative tracings are shown from platelets activated with the indicated concentrations of: ( A ) thrombin (1–100 nM), ( C ) AYPGKF (0.15–2 mM), or ( E ) 3 µM thapsigargin (TG). Quantitation of the change in peak Ca 2+ mobilization in platelets stimulated with: ( B ) thrombin, ( D ) AYPGKF, or ( F ) thapsigargin. The results are the mean (± SD) of three independent experiments (* p <0.05).

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: Fura 2-loaded wild type (black line) and PAR3 −/− (gray line) platelets were resuspended in Ca 2+ -free medium (0.1 mM EGTA was added at the time of experiment). Representative tracings are shown from platelets activated with the indicated concentrations of: ( A ) thrombin (1–100 nM), ( C ) AYPGKF (0.15–2 mM), or ( E ) 3 µM thapsigargin (TG). Quantitation of the change in peak Ca 2+ mobilization in platelets stimulated with: ( B ) thrombin, ( D ) AYPGKF, or ( F ) thapsigargin. The results are the mean (± SD) of three independent experiments (* p <0.05).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Quantitation Assay

The HEK293 cells were transfected with: ( A ) PAR4-Luc (1 µg) and PAR3-GFP (0–2.5 µg), ( B ) PAR3-Luc (1 µg) and PAR3-GFP (0–2.5 µg), or ( C ) PAR4-Luc (1 µg) and PAR4-GFP (0–2.5 µg). As a control experiment, the HEK293 cells were transfected with: ( D ) PAR3-Luc (1 µg) and rho-GFP (0–0.12 µg), or ( E ) PAR4-Luc (1 µg) and rho-GFP (0–0.12 µg). Forty-eight hours post-transfection, the cells were analyzed for GFP expression, Luc expression, and BRET. The curves were plotted as the ratio of GFP to Luc and all points from 3–6 independent experiments were analyzed by global fit to a hyperbolic or linear curve. The surface expression of PAR3 and PAR4 in the HEK293 cells was determined by flow cytometry. ( G ) V5-PAR4-GFP and V5-PAR3-GFP were detected with a V5 tag antibody conjugated to Alexa Fluor 647. ( H ) HA-PAR4-LUC and HA-PAR3-LUC were detected with a HA tag antibody conjugated to Alexa Fluor 647. The results are the mean (± SD) of two independent experiments. The number of PAR4 and PAR3 molecules on the HEK293 cells surface is calculated from the V5 or HA antibody standard curve using quantitative flow cytometry ( F ).

Journal: PLoS ONE

Article Title: Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

doi: 10.1371/journal.pone.0055740

Figure Lengend Snippet: The HEK293 cells were transfected with: ( A ) PAR4-Luc (1 µg) and PAR3-GFP (0–2.5 µg), ( B ) PAR3-Luc (1 µg) and PAR3-GFP (0–2.5 µg), or ( C ) PAR4-Luc (1 µg) and PAR4-GFP (0–2.5 µg). As a control experiment, the HEK293 cells were transfected with: ( D ) PAR3-Luc (1 µg) and rho-GFP (0–0.12 µg), or ( E ) PAR4-Luc (1 µg) and rho-GFP (0–0.12 µg). Forty-eight hours post-transfection, the cells were analyzed for GFP expression, Luc expression, and BRET. The curves were plotted as the ratio of GFP to Luc and all points from 3–6 independent experiments were analyzed by global fit to a hyperbolic or linear curve. The surface expression of PAR3 and PAR4 in the HEK293 cells was determined by flow cytometry. ( G ) V5-PAR4-GFP and V5-PAR3-GFP were detected with a V5 tag antibody conjugated to Alexa Fluor 647. ( H ) HA-PAR4-LUC and HA-PAR3-LUC were detected with a HA tag antibody conjugated to Alexa Fluor 647. The results are the mean (± SD) of two independent experiments. The number of PAR4 and PAR3 molecules on the HEK293 cells surface is calculated from the V5 or HA antibody standard curve using quantitative flow cytometry ( F ).

Article Snippet: The cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library.

Techniques: Transfection, Control, Expressing, Flow Cytometry

a) Simplified phylogeny of animals shows that acoel brains are likely intermediate between cnidarian diffuse nets and the centralized brains of typical bilaterians. b) Photograph of juvenile Hofstenia miamia . c) Staining with voltage dye reveals a superficial network of dense neuropil (blue arrow) that extends into a sparser posterior nerve net (green arrow). d) Close-up view of neuropil stained sparsely with tubulin dye (orange) reveals that the neuropil (orange) contains many neurites running in parallel, with cellular clusters (cyan) interspersed between neurite bundles. Sensory neurons (likely clusters of H1 cells; bright orange) are set within many of these patches. e) Cross-section of brain stained with a Par3 antibody reveals that the brain has two layers: superficial neuropil, and deeper cell bodies that project outward. f) Staining with an ERK antibody (z-projected segmentation overlaid) shows that brain interneurons can be multipolar, with a central cell body generating multiple neurites. g) Cross-section of brain stained with an antibody against β-catenin reveals another sensory neuron class (possibly H2 ) with two projections that innervate brain neuropil. h) Electron microscopy cross-section shows the fine organization of the brain, confirming the relative configuration of tissue types within the head. The superficial neuropil (previously ‘layer 1’) is visible immediately beneath the skin, while neural cell bodies (previously ‘layer 2’) lie deeper in the tissue, internal to body wall muscle (green). Together, these layers compose the brain. i) Electron microscopy close-up of the brain shows dense neuropil; the box is a 6.7×6.7µm square. j) Segmenting neural projections within the highlighted box in (i) reveals over 400 neurites in a single section of neuropil. k) Segmentation of cellular clusters within neuropil allows quantification of brain structure and its variability. l) Quantifying the numbers of cellular clusters across brains reveals that, although cluster numbers increase with age (i.e. days after hatching) and size (i.e. head width, a good proxy for overall body size ), worms vary widely in how many clusters they possess. Linear regression p<0.0001, n=49. Scale bars: 200µm (c), 50µm (d,e), 20µm (f,g), 10µm (h).

Journal: bioRxiv

Article Title: Distributed neural computation and the evolution of the first brains

doi: 10.1101/2025.10.03.680388

Figure Lengend Snippet: a) Simplified phylogeny of animals shows that acoel brains are likely intermediate between cnidarian diffuse nets and the centralized brains of typical bilaterians. b) Photograph of juvenile Hofstenia miamia . c) Staining with voltage dye reveals a superficial network of dense neuropil (blue arrow) that extends into a sparser posterior nerve net (green arrow). d) Close-up view of neuropil stained sparsely with tubulin dye (orange) reveals that the neuropil (orange) contains many neurites running in parallel, with cellular clusters (cyan) interspersed between neurite bundles. Sensory neurons (likely clusters of H1 cells; bright orange) are set within many of these patches. e) Cross-section of brain stained with a Par3 antibody reveals that the brain has two layers: superficial neuropil, and deeper cell bodies that project outward. f) Staining with an ERK antibody (z-projected segmentation overlaid) shows that brain interneurons can be multipolar, with a central cell body generating multiple neurites. g) Cross-section of brain stained with an antibody against β-catenin reveals another sensory neuron class (possibly H2 ) with two projections that innervate brain neuropil. h) Electron microscopy cross-section shows the fine organization of the brain, confirming the relative configuration of tissue types within the head. The superficial neuropil (previously ‘layer 1’) is visible immediately beneath the skin, while neural cell bodies (previously ‘layer 2’) lie deeper in the tissue, internal to body wall muscle (green). Together, these layers compose the brain. i) Electron microscopy close-up of the brain shows dense neuropil; the box is a 6.7×6.7µm square. j) Segmenting neural projections within the highlighted box in (i) reveals over 400 neurites in a single section of neuropil. k) Segmentation of cellular clusters within neuropil allows quantification of brain structure and its variability. l) Quantifying the numbers of cellular clusters across brains reveals that, although cluster numbers increase with age (i.e. days after hatching) and size (i.e. head width, a good proxy for overall body size ), worms vary widely in how many clusters they possess. Linear regression p<0.0001, n=49. Scale bars: 200µm (c), 50µm (d,e), 20µm (f,g), 10µm (h).

Article Snippet: Primary antibodies used: Par3 (St. John’s Laboratory #STJ94951, 1:200), pERK (Cell Signaling Technologies #4370T, 1:200) , FMRFamide (EMDMillipore #AB15348, 1:1000) .

Techniques: Staining, Electron Microscopy