mouse anti-par3 Search Results


mouse p4a1 anti par 3  (Developmental Studies Hybridoma Bank)
91
Developmental Studies Hybridoma Bank mouse p4a1 anti par 3
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/mouse+anti-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, 2026-07
91/100 stars
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anti par3 sc 5598 anti par4  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology anti par3 sc 5598 anti par4
Anti Par3 Sc 5598 Anti Par4, 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/mouse+anti-par3/10__1074_slash_jbc__ra118__003491-165-4-29?v=Santa+Cruz+Biotechnology
Average 93 stars, based on 1 article reviews
anti par3 sc 5598 anti par4 - by Bioz Stars, 2026-07
93/100 stars
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rabbit anti pard3a  (Proteintech)
93
Proteintech rabbit anti pard3a
Rabbit Anti Pard3a, 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/mouse+anti-par3/pmc08129141-391-129-131?v=Proteintech
Average 93 stars, based on 1 article reviews
rabbit anti pard3a - by Bioz Stars, 2026-07
93/100 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/mouse+anti-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, 2026-07
93/100 stars
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anti par 3 mouse monoclonal  (Rockland Immunochemicals)
93
Rockland Immunochemicals anti par 3 mouse monoclonal
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).
Anti Par 3 Mouse Monoclonal, supplied by Rockland Immunochemicals, 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/mouse+anti-par3/pmc02915941-87-8-21?v=Rockland+Immunochemicals
Average 93 stars, based on 1 article reviews
anti par 3 mouse monoclonal - by Bioz Stars, 2026-07
93/100 stars
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goat anti par 3  (Santa Cruz Biotechnology)
99
Santa Cruz Biotechnology goat anti par 3
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).
Goat Anti Par 3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+anti-par3/pm25788872-60-55-58?v=Santa+Cruz+Biotechnology
Average 99 stars, based on 1 article reviews
goat anti par 3 - by Bioz Stars, 2026-07
99/100 stars
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rabbit anti par3  (Novus Biologicals)
94
Novus Biologicals rabbit anti par3
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).
Rabbit Anti 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/mouse+anti-par3/pmc05703611-232-129-131?v=Novus+Biologicals
Average 94 stars, based on 1 article reviews
rabbit anti par3 - by Bioz Stars, 2026-07
94/100 stars
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rabbit anti pard3  (Novus Biologicals)
94
Novus Biologicals rabbit anti pard3
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).
Rabbit Anti Pard3, 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/mouse+anti-par3/bio_rxiv__2024__06__30__601407-256-22-24?v=Novus+Biologicals
Average 94 stars, based on 1 article reviews
rabbit anti pard3 - by Bioz Stars, 2026-07
94/100 stars
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par antibody  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology par 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).
Par Antibody, supplied by Santa Cruz Biotechnology, 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/mouse+anti-par3/pm12944324-83-13-28?v=Santa+Cruz+Biotechnology
Average 94 stars, based on 1 article reviews
par antibody - by Bioz Stars, 2026-07
94/100 stars
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lpar3 antibody - middle region (oaga01142)  (Aviva Systems)
N/A
Rabbit Polyclonal antibody to EDG7 (lysophosphatidic acid receptor 3); This gene encodes a member of the G protein-coupled receptor family, as well as the EDG family of proteins. This protein functions as a cellular receptor
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pard3 antibody (oala05828)  (Aviva Systems)
N/A
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lpar3 antibody (oala00007)  (Aviva Systems)
N/A
EDG7, a Lysophospholipid/Lysosphingolipid Receptor, was identified by RT-PCR from Jurkat T-cells. It is activated by unsaturated lysophosphatidic acid LPA.
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Image Search Results


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: