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    Name:
    Recombinant Protein Purification Handbook
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    Catalog Number:
    ge18-1142-75
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    Structured Review

    Millipore recombinant protein

    https://www.bioz.com/result/recombinant protein/product/Millipore
    Average 99 stars, based on 800 article reviews
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    Related Articles

    Clone Assay:

    Article Title: Expression of the Floral Repressor miRNA156 is Positively Regulated by the AGAMOUS-like Proteins AGL15 and AGL18
    Article Snippet: .. Recombinant protein production and purification For production of His-fused proteins, the open reading frame (ORF) of Arabidopsis AGL15 or AGL18 was cloned into the pET21a vector (EMD Biosciences, USA). .. The resulting construct was introduced into E. coli BL21.

    Flow Cytometry:

    Article Title: Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers
    Article Snippet: .. The spent culture medium containing recombinant protein was filtered (0.65 + 0.45 μm Sartobran 300 capsule, PALL) and concentrated by tangential flow filtration (PelliconTM -2 system, Millipore) with a 30-kDa PLCGC filter. ..

    Filtration:

    Article Title: Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers
    Article Snippet: .. The spent culture medium containing recombinant protein was filtered (0.65 + 0.45 μm Sartobran 300 capsule, PALL) and concentrated by tangential flow filtration (PelliconTM -2 system, Millipore) with a 30-kDa PLCGC filter. ..

    Chromatography:

    Article Title: Structural basis for the core-mannan biosynthesis of cell wall fungal-type galactomannan in Aspergillusfumigatus.
    Article Snippet: .. CmsA expression in E. coli and recombinant protein purification The catalytic domain of A. fumigatus CmsA/Ktr4 was expressed in an Escherichia coli system and purified by Ni-affinity chromatography as described previously (30). .. Protein concentration was determined using a Qubit Protein Assay Kit (Thermo Fisher Scientific, USA) while purity and molecular weight were analyzed by SDS-PAGE.

    Purification:

    Article Title: Structural basis for the core-mannan biosynthesis of cell wall fungal-type galactomannan in Aspergillusfumigatus.
    Article Snippet: .. CmsA expression in E. coli and recombinant protein purification The catalytic domain of A. fumigatus CmsA/Ktr4 was expressed in an Escherichia coli system and purified by Ni-affinity chromatography as described previously (30). .. Protein concentration was determined using a Qubit Protein Assay Kit (Thermo Fisher Scientific, USA) while purity and molecular weight were analyzed by SDS-PAGE.

    Article Title: Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M
    Article Snippet: .. Confirmation of recombinant protein The purified recombinant protein was electrophoresed on 12.5 % SDS-PAGE and was subsequently transferred to nitrocellulose membrane (Millipore, Bedford, USA). .. The membrane was blocked with 3% blocking buffer (PBS-1X containing 3% skim milk) at room temperature for 12 h and washed three times with washing buffer (PBS-1X containing 0.05% Tween 20).

    Article Title: Expression of the Floral Repressor miRNA156 is Positively Regulated by the AGAMOUS-like Proteins AGL15 and AGL18
    Article Snippet: .. Recombinant protein production and purification For production of His-fused proteins, the open reading frame (ORF) of Arabidopsis AGL15 or AGL18 was cloned into the pET21a vector (EMD Biosciences, USA). .. The resulting construct was introduced into E. coli BL21.

    Article Title: Detection of Bcl-2 family member Bcl-G in mouse tissues using new monoclonal antibodies
    Article Snippet: .. The purified recombinant protein was then concentrated using centrifugal filter units (10 000 MWCO, Millipore, Bedford, MA, USA) and used for immunisation. .. Immunisation Wistar rats were immunised by injection of 100 μ g of purified His-tagged mBcl-G protein.

    Article Title: Development of a sensitive Luminex xMAP-based microsphere immunoassay for specific detection of Iris yellow spot virus
    Article Snippet: .. For the first subcutaneous injection, 0.8 mg purified recombinant N protein in 1 ml phosphate-buffered saline (PBS) was emulsified with an equal volume of complete Freund’s adjuvant (Sigma-Aldrich). .. Subsequently, the recombinant protein (0.5 mg in 1 ml PBS) emulsified with an equal volume of incomplete Freund’s adjuvant (Sigma-Aldrich) was administered at 3-week interval for three times.

    Protein Purification:

    Article Title: Structural basis for the core-mannan biosynthesis of cell wall fungal-type galactomannan in Aspergillusfumigatus.
    Article Snippet: .. CmsA expression in E. coli and recombinant protein purification The catalytic domain of A. fumigatus CmsA/Ktr4 was expressed in an Escherichia coli system and purified by Ni-affinity chromatography as described previously (30). .. Protein concentration was determined using a Qubit Protein Assay Kit (Thermo Fisher Scientific, USA) while purity and molecular weight were analyzed by SDS-PAGE.

    Plasmid Preparation:

    Article Title: Equine Arteritis Virus Uses Equine CXCL16 as an Entry Receptor
    Article Snippet: .. In order to express recombinant protein, the plasmid was transformed into E. coli BL21(DE3) cells (EMD Millipore Novagen, Temecula, CA). ..

    Article Title: Expression of the Floral Repressor miRNA156 is Positively Regulated by the AGAMOUS-like Proteins AGL15 and AGL18
    Article Snippet: .. Recombinant protein production and purification For production of His-fused proteins, the open reading frame (ORF) of Arabidopsis AGL15 or AGL18 was cloned into the pET21a vector (EMD Biosciences, USA). .. The resulting construct was introduced into E. coli BL21.

    Expressing:

    Article Title: Structural basis for the core-mannan biosynthesis of cell wall fungal-type galactomannan in Aspergillusfumigatus.
    Article Snippet: .. CmsA expression in E. coli and recombinant protein purification The catalytic domain of A. fumigatus CmsA/Ktr4 was expressed in an Escherichia coli system and purified by Ni-affinity chromatography as described previously (30). .. Protein concentration was determined using a Qubit Protein Assay Kit (Thermo Fisher Scientific, USA) while purity and molecular weight were analyzed by SDS-PAGE.

    Western Blot:

    Article Title: Expression and purification of turkey coronavirus nucleocapsid protein in Escherichia coli
    Article Snippet: .. Identity of the recombinant N protein was confirmed by SDS-PAGE of fractions eluted from the column and Western blotting analysis of electrotransferred protein on nitrocellulose membrane (Millipore) with reagent specific to histidine tag, horseradish peroxidase-conjugated nickel-NTA (Qiagen). .. 2.6 SDS-polyacrylamide gel electrophoresis and Western immunoblotting The samples were solubilized in sample buffer containing 62.5 mM Tris–HCl, pH 6.8, 1% SDS, 10% glycerol, 0.001% bromophenol blue, and 1% 2-mercaptoethanol and boiled for 5 min.

    Injection:

    Article Title: Development of a sensitive Luminex xMAP-based microsphere immunoassay for specific detection of Iris yellow spot virus
    Article Snippet: .. For the first subcutaneous injection, 0.8 mg purified recombinant N protein in 1 ml phosphate-buffered saline (PBS) was emulsified with an equal volume of complete Freund’s adjuvant (Sigma-Aldrich). .. Subsequently, the recombinant protein (0.5 mg in 1 ml PBS) emulsified with an equal volume of incomplete Freund’s adjuvant (Sigma-Aldrich) was administered at 3-week interval for three times.

    Recombinant:

    Article Title: Expression and purification of turkey coronavirus nucleocapsid protein in Escherichia coli
    Article Snippet: .. Identity of the recombinant N protein was confirmed by SDS-PAGE of fractions eluted from the column and Western blotting analysis of electrotransferred protein on nitrocellulose membrane (Millipore) with reagent specific to histidine tag, horseradish peroxidase-conjugated nickel-NTA (Qiagen). .. 2.6 SDS-polyacrylamide gel electrophoresis and Western immunoblotting The samples were solubilized in sample buffer containing 62.5 mM Tris–HCl, pH 6.8, 1% SDS, 10% glycerol, 0.001% bromophenol blue, and 1% 2-mercaptoethanol and boiled for 5 min.

    Article Title: Structural basis for the core-mannan biosynthesis of cell wall fungal-type galactomannan in Aspergillusfumigatus.
    Article Snippet: .. CmsA expression in E. coli and recombinant protein purification The catalytic domain of A. fumigatus CmsA/Ktr4 was expressed in an Escherichia coli system and purified by Ni-affinity chromatography as described previously (30). .. Protein concentration was determined using a Qubit Protein Assay Kit (Thermo Fisher Scientific, USA) while purity and molecular weight were analyzed by SDS-PAGE.

    Article Title: Equine Arteritis Virus Uses Equine CXCL16 as an Entry Receptor
    Article Snippet: .. In order to express recombinant protein, the plasmid was transformed into E. coli BL21(DE3) cells (EMD Millipore Novagen, Temecula, CA). ..

    Article Title: Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M
    Article Snippet: .. Confirmation of recombinant protein The purified recombinant protein was electrophoresed on 12.5 % SDS-PAGE and was subsequently transferred to nitrocellulose membrane (Millipore, Bedford, USA). .. The membrane was blocked with 3% blocking buffer (PBS-1X containing 3% skim milk) at room temperature for 12 h and washed three times with washing buffer (PBS-1X containing 0.05% Tween 20).

    Article Title: Expression of the Floral Repressor miRNA156 is Positively Regulated by the AGAMOUS-like Proteins AGL15 and AGL18
    Article Snippet: .. Recombinant protein production and purification For production of His-fused proteins, the open reading frame (ORF) of Arabidopsis AGL15 or AGL18 was cloned into the pET21a vector (EMD Biosciences, USA). .. The resulting construct was introduced into E. coli BL21.

    Article Title: Detection of Bcl-2 family member Bcl-G in mouse tissues using new monoclonal antibodies
    Article Snippet: .. The purified recombinant protein was then concentrated using centrifugal filter units (10 000 MWCO, Millipore, Bedford, MA, USA) and used for immunisation. .. Immunisation Wistar rats were immunised by injection of 100 μ g of purified His-tagged mBcl-G protein.

    Article Title: Development of a sensitive Luminex xMAP-based microsphere immunoassay for specific detection of Iris yellow spot virus
    Article Snippet: .. For the first subcutaneous injection, 0.8 mg purified recombinant N protein in 1 ml phosphate-buffered saline (PBS) was emulsified with an equal volume of complete Freund’s adjuvant (Sigma-Aldrich). .. Subsequently, the recombinant protein (0.5 mg in 1 ml PBS) emulsified with an equal volume of incomplete Freund’s adjuvant (Sigma-Aldrich) was administered at 3-week interval for three times.

    Article Title: Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers
    Article Snippet: .. The spent culture medium containing recombinant protein was filtered (0.65 + 0.45 μm Sartobran 300 capsule, PALL) and concentrated by tangential flow filtration (PelliconTM -2 system, Millipore) with a 30-kDa PLCGC filter. ..

    Transformation Assay:

    Article Title: Equine Arteritis Virus Uses Equine CXCL16 as an Entry Receptor
    Article Snippet: .. In order to express recombinant protein, the plasmid was transformed into E. coli BL21(DE3) cells (EMD Millipore Novagen, Temecula, CA). ..

    SDS Page:

    Article Title: Expression and purification of turkey coronavirus nucleocapsid protein in Escherichia coli
    Article Snippet: .. Identity of the recombinant N protein was confirmed by SDS-PAGE of fractions eluted from the column and Western blotting analysis of electrotransferred protein on nitrocellulose membrane (Millipore) with reagent specific to histidine tag, horseradish peroxidase-conjugated nickel-NTA (Qiagen). .. 2.6 SDS-polyacrylamide gel electrophoresis and Western immunoblotting The samples were solubilized in sample buffer containing 62.5 mM Tris–HCl, pH 6.8, 1% SDS, 10% glycerol, 0.001% bromophenol blue, and 1% 2-mercaptoethanol and boiled for 5 min.

    Article Title: Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M
    Article Snippet: .. Confirmation of recombinant protein The purified recombinant protein was electrophoresed on 12.5 % SDS-PAGE and was subsequently transferred to nitrocellulose membrane (Millipore, Bedford, USA). .. The membrane was blocked with 3% blocking buffer (PBS-1X containing 3% skim milk) at room temperature for 12 h and washed three times with washing buffer (PBS-1X containing 0.05% Tween 20).

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  • 93
    Millipore recombinant hca59 protein
    Expression, purification and Western blot analysis of rHCA59 protein. Lane M: standard protein molecular weight marker. a rHCA59 protein was induced with 1 mM IPTG. Lane 1: expression of recombinant protein rHCA59 vector before purification; Lane 2: purified rHCA59 protein. b Western blot of rHCA59 protein. Lane 1: recombinant protein <t>HCA59</t> was recognized by goat anti- H. contortus sera; Lane 2: membrane incubated with normal goat sera (as control). c Western blot of total HcESPs. Lane 1: HcESPs were detected by rat anti-rHCA59 protein antibodies; Lane 2: membrane incubated with normal rat sera (as control)
    Recombinant Hca59 Protein, supplied by Millipore, 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/result/recombinant hca59 protein/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant hca59 protein - by Bioz Stars, 2020-11
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    85
    Millipore recombinant opticin
    <t>Opticin</t> inhibits collagen type I and Matrigel TM -stimulated angiogenesis in vitro . Opticin (250 n m ) significantly decreased FGF-2-stimulated HUVEC capillary morphogenesis in collagen type I ( A ) (*, p
    Recombinant Opticin, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant opticin/product/Millipore
    Average 85 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    recombinant opticin - by Bioz Stars, 2020-11
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    89
    Millipore recombinant ck2
    Rodent Par-4 phosphorylation by <t>CK2</t> prevents caspase-mediated Par-4 cleavage. ( a ) PC-3 cells transfected with the GFP-tagged constructs wild-type rat Par-4, 124A/D, 223A/D, 124A223A or 124D223D mutants of Par-4 were treated with TRAIL (500 ng/ml, 3 h) in the presence or absence of Z-VAD (15 μ M). Par-4 cleavage was determined by western blotting using anti-GFP antibody. Hsp90 was used as a loading control. ( b ) Wild-type Par-4, 124A/D, 223A/D, 124D223D and 124A223A mutant proteins (GFP tagged) were produced in vitro by a TNT RRL system. Recombinant caspase-8 (upper panel) or caspase-3 and -7 (Casp3, Casp7, lower panel) were incubated with the indicated Par-4 proteins for 3 h at 37 °C, and Par-4 cleavage was assessed by western blotting using GFP antibody. ( c ) Caspase assay was performed on pre-phosphorylated rat GST-Par-4 ( in vitro kinase assay using RecCK2 as described above Figure 2 ), and cleaved Par-4 was detected by immunoblot. *NS: non-specific bands are probably due to the presence of recombinant Par-4 degraded forms. To note: cleaved GST Par-4 migrates at lower molecular weight than cleaved GFP-Par-4, given GST tag is located in the N-terminus of Par-4 and not on C-terminus as the GFP-tag. ( d–f ) PC-3 cells transfected with Par-4 wild-type or FLAG-tagged Par-4 (124–332) were treated as above with TRAIL and analyzed for apoptosis by Hoechst staining ( d ) or FACS analysis of the caspase-3 activity (FLICA) ( e ) and by immunoblotting analysis of caspase-8 and PARP cleavage ( f ). Bars represent the mean±S.D. of at least three independent experiments. Flag-Par-4 (124–332) and GFP were revealed in the same membrane using corresponding antibodies (lower panel). Endogenous Par-4 was used as a loading control. * P
    Recombinant Ck2, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant ck2/product/Millipore
    Average 89 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
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    85
    Millipore recombinant myd88 5
    Generation of <t>MyD88-5</t> knockout mice and protection of their hippocampal neurons from OGD-induced cell death. (A) Schematic of the MyD88-5 locus, targeting vector, and predicted structure of the mutated locus. Exon numbers are indicated. The probe used for Southern blotting is shown in red. Restriction enzyme sites and predicted sizes of the digests are shown. Sp, SphI; Neo, neomycin resistance gene; DTX, diphtheria toxin gene. (B) Southern blot analysis of genomic DNA from MyD88-5 wild-type, hemizygous-deficient, and null mice. (C) Western blot analysis of MyD88-5 expression in wild-type, hemizygous-deficient, and null mice. 50 μg of brain lysates were subjected to SDS-PAGE. Affinity-purified chicken anti–MyD88-5 antibody was used for detection. (D) Propidium iodide staining of dead neurons in hippocampal slices at the start of the OGD period (basal), after OGD (OGD), or the same period without OGD treatment (sham), and after exposure to 1 mM NMDA to induce a maximal cell death (Max). Hippocampi illustrated were from MyD88-5 +/− (Het) or MyD88-5 −/− mice (KO). Bar, 1 mm. (E) Quantitative evaluation of cell death after sham or OGD treatment in MyD88-5 +/+ (sham, n = 6; OGD, n = 13) MyD88-5 +/− (sham, n = 11; OGD, n = 22) and MyD88-5 −/− (sham, n = 5; OGD, n = 29) slices. P
    Recombinant Myd88 5, supplied by Millipore, 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/result/recombinant myd88 5/product/Millipore
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Expression, purification and Western blot analysis of rHCA59 protein. Lane M: standard protein molecular weight marker. a rHCA59 protein was induced with 1 mM IPTG. Lane 1: expression of recombinant protein rHCA59 vector before purification; Lane 2: purified rHCA59 protein. b Western blot of rHCA59 protein. Lane 1: recombinant protein HCA59 was recognized by goat anti- H. contortus sera; Lane 2: membrane incubated with normal goat sera (as control). c Western blot of total HcESPs. Lane 1: HcESPs were detected by rat anti-rHCA59 protein antibodies; Lane 2: membrane incubated with normal rat sera (as control)

    Journal: Parasites & Vectors

    Article Title: Hepatocellular carcinoma-associated antigen 59 of Haemonchus contortus modulates the functions of PBMCs and the differentiation and maturation of monocyte-derived dendritic cells of goats in vitro

    doi: 10.1186/s13071-019-3375-1

    Figure Lengend Snippet: Expression, purification and Western blot analysis of rHCA59 protein. Lane M: standard protein molecular weight marker. a rHCA59 protein was induced with 1 mM IPTG. Lane 1: expression of recombinant protein rHCA59 vector before purification; Lane 2: purified rHCA59 protein. b Western blot of rHCA59 protein. Lane 1: recombinant protein HCA59 was recognized by goat anti- H. contortus sera; Lane 2: membrane incubated with normal goat sera (as control). c Western blot of total HcESPs. Lane 1: HcESPs were detected by rat anti-rHCA59 protein antibodies; Lane 2: membrane incubated with normal rat sera (as control)

    Article Snippet: The recombinant HCA59 protein was separated on 12% SDS-PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon-PSQ, Millipore, Billerica, MA, USA) using a semi-dry system (Novablot, Hoefer, USA) in transfer buffer (39 mM glycine, 48 mM Tris, 20% methanol, 0.0375% SDS) at 1.5 mA/cm2 for 25 min. Next, the membrane was blocked with 5% (w/v) skim milk powder in TBST (TBS with 0.5% Tween-20) at 37 °C for 2 h, washed three times with TBST and incubated with anti-H. contortus sera from goats (1:300 dilutions) as the first antibody in the treatment group or normal goat sera in the negative control group (1:300 dilutions) for one night at 4 °C.

    Techniques: Expressing, Purification, Western Blot, Molecular Weight, Marker, Recombinant, Plasmid Preparation, Incubation

    Opticin inhibits collagen type I and Matrigel TM -stimulated angiogenesis in vitro . Opticin (250 n m ) significantly decreased FGF-2-stimulated HUVEC capillary morphogenesis in collagen type I ( A ) (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness *

    doi: 10.1074/jbc.M111.331157

    Figure Lengend Snippet: Opticin inhibits collagen type I and Matrigel TM -stimulated angiogenesis in vitro . Opticin (250 n m ) significantly decreased FGF-2-stimulated HUVEC capillary morphogenesis in collagen type I ( A ) (*, p

    Article Snippet: Cell Proliferation Assays Using Bromodeoxyuridine (BrdU) Incorporations The effects of recombinant opticin on EC proliferation were tested using BrdU incorporation assays (Calbiochem®, Nottingham, UK).

    Techniques: In Vitro

    Effects of opticin in CAM ex vivo model. A , representative images showing the effects of a pellet without FGF-2 or opticin ( left image), and pellets containing FGF-2 with or without opticin. Asterisk indicates the site of application of the pellet. Under FGF-2 stimulation ( middle image), newly formed vessels converge toward where the growth factor-containing pellet was placed ( arrows ). B , pellets containing growth factors i.e. FGF-2, VEGF 165 , and BBE, with or without opticin, were applied on the CAM, and the angiogenic response was quantified at day 10. Addition of opticin in combination with FGF-2 ( n = 8), VEGF 165 ( n = 12), and BBE ( n = 12) significantly reduced their angiogenic drive on the CAM (*, p = 0.01, p = 0.04, and p

    Journal: The Journal of Biological Chemistry

    Article Title: Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness *

    doi: 10.1074/jbc.M111.331157

    Figure Lengend Snippet: Effects of opticin in CAM ex vivo model. A , representative images showing the effects of a pellet without FGF-2 or opticin ( left image), and pellets containing FGF-2 with or without opticin. Asterisk indicates the site of application of the pellet. Under FGF-2 stimulation ( middle image), newly formed vessels converge toward where the growth factor-containing pellet was placed ( arrows ). B , pellets containing growth factors i.e. FGF-2, VEGF 165 , and BBE, with or without opticin, were applied on the CAM, and the angiogenic response was quantified at day 10. Addition of opticin in combination with FGF-2 ( n = 8), VEGF 165 ( n = 12), and BBE ( n = 12) significantly reduced their angiogenic drive on the CAM (*, p = 0.01, p = 0.04, and p

    Article Snippet: Cell Proliferation Assays Using Bromodeoxyuridine (BrdU) Incorporations The effects of recombinant opticin on EC proliferation were tested using BrdU incorporation assays (Calbiochem®, Nottingham, UK).

    Techniques: Chick Chorioallantoic Membrane Assay, Ex Vivo

    Opticin inhibits interactions between integrins and collagens/laminin. A , binding studies between opticin and various substrates revealed that it interacted strongly with collagen type I (●), collagen type II (○), and laminin (Δ), and more weakly to collagen type IV (▾). However, no binding was observed to vitronectin (■) or fibronectin (□). B , binding assays showed that both α 1 β 1 and α 2 β 1 integrins interacted with collagen type I strongly (▾ and Δ, respectively), but no binding to opticin was observed for either integrins (● and ○, respectively). C , inhibition studies using a fixed concentration of collagen type I preincubated with increasing opticin concentrations revealed that opticin inhibited collagen type I binding to both α 1 β 1 and α 2 β 1 integrins (● and ○, respectively). This inhibition was significant for both integrins at opticin concentrations as low as 1.75 n m ( p = 0.02 and p = 0.002 for α 1 β 1 and α 2 β 1 integrins, respectively), corresponding to a 1:1 molar ratio between collagen type I and opticin; background absorbance level is indicated by a dashed line .

    Journal: The Journal of Biological Chemistry

    Article Title: Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness *

    doi: 10.1074/jbc.M111.331157

    Figure Lengend Snippet: Opticin inhibits interactions between integrins and collagens/laminin. A , binding studies between opticin and various substrates revealed that it interacted strongly with collagen type I (●), collagen type II (○), and laminin (Δ), and more weakly to collagen type IV (▾). However, no binding was observed to vitronectin (■) or fibronectin (□). B , binding assays showed that both α 1 β 1 and α 2 β 1 integrins interacted with collagen type I strongly (▾ and Δ, respectively), but no binding to opticin was observed for either integrins (● and ○, respectively). C , inhibition studies using a fixed concentration of collagen type I preincubated with increasing opticin concentrations revealed that opticin inhibited collagen type I binding to both α 1 β 1 and α 2 β 1 integrins (● and ○, respectively). This inhibition was significant for both integrins at opticin concentrations as low as 1.75 n m ( p = 0.02 and p = 0.002 for α 1 β 1 and α 2 β 1 integrins, respectively), corresponding to a 1:1 molar ratio between collagen type I and opticin; background absorbance level is indicated by a dashed line .

    Article Snippet: Cell Proliferation Assays Using Bromodeoxyuridine (BrdU) Incorporations The effects of recombinant opticin on EC proliferation were tested using BrdU incorporation assays (Calbiochem®, Nottingham, UK).

    Techniques: Binding Assay, Inhibition, Concentration Assay

    Opticin disrupts HUVEC morphology when spread on collagen types I, II, and IV and laminin. A , opticin was added at 250 n m in solution to HUVECs spread on plates coated with collagen types I, II, and IV, and laminin, vitronectin, or fibronectin. Visualization of stress fibers (actin in red ), focal adhesions (vinculin in green ), and nuclei (in blue ) showed that in absence of opticin, HUVECs spread on all these matrices; focal adhesions and actin stress fibers were clearly visible ( top panel ). However, after exposure to opticin the cells retracted: the stress fibers and focal adhesion disappeared when spread on collagen types I, II, and IV and laminin ( bottom panel ). The cell morphology was unaltered by opticin on vitronectin or fibronectin ( scale bar , 50 μm). B , quantification showed that opticin disrupted HUVEC cytoskeleton in a concentration-dependent manner on collagen type I (●), collagen type II (○), collagen type IV (Δ), and laminin (▾) but not on vitronectin (■) or fibronectin (□); significant effects were observed at concentrations as low as 10 n m on collagen type I (*, p = 0.017). C , visualization of actin stress fibers (in red ) and focal adhesions (vinculin and paxillin in green ) showed that the discrete localization of both vinculin ( top panel ) and paxillin ( bottom panel ) in focal adhesions was lost when HUVEC spread on collagen type I were exposed to opticin at 250 n m ( scale bar , 10 μm). D , when the effects of opticin and another SLRP family member, decorin, were compared, decorin had no effect, whereas opticin significantly disrupted HUVEC morphology (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness *

    doi: 10.1074/jbc.M111.331157

    Figure Lengend Snippet: Opticin disrupts HUVEC morphology when spread on collagen types I, II, and IV and laminin. A , opticin was added at 250 n m in solution to HUVECs spread on plates coated with collagen types I, II, and IV, and laminin, vitronectin, or fibronectin. Visualization of stress fibers (actin in red ), focal adhesions (vinculin in green ), and nuclei (in blue ) showed that in absence of opticin, HUVECs spread on all these matrices; focal adhesions and actin stress fibers were clearly visible ( top panel ). However, after exposure to opticin the cells retracted: the stress fibers and focal adhesion disappeared when spread on collagen types I, II, and IV and laminin ( bottom panel ). The cell morphology was unaltered by opticin on vitronectin or fibronectin ( scale bar , 50 μm). B , quantification showed that opticin disrupted HUVEC cytoskeleton in a concentration-dependent manner on collagen type I (●), collagen type II (○), collagen type IV (Δ), and laminin (▾) but not on vitronectin (■) or fibronectin (□); significant effects were observed at concentrations as low as 10 n m on collagen type I (*, p = 0.017). C , visualization of actin stress fibers (in red ) and focal adhesions (vinculin and paxillin in green ) showed that the discrete localization of both vinculin ( top panel ) and paxillin ( bottom panel ) in focal adhesions was lost when HUVEC spread on collagen type I were exposed to opticin at 250 n m ( scale bar , 10 μm). D , when the effects of opticin and another SLRP family member, decorin, were compared, decorin had no effect, whereas opticin significantly disrupted HUVEC morphology (*, p

    Article Snippet: Cell Proliferation Assays Using Bromodeoxyuridine (BrdU) Incorporations The effects of recombinant opticin on EC proliferation were tested using BrdU incorporation assays (Calbiochem®, Nottingham, UK).

    Techniques: Concentration Assay

    Opticin inhibits capillary morphogenesis, promotes capillary network regression, and inhibits EC invasion in collagen gels. Three different types of EC were used: BAECs ( black bars ), HRECs ( white bars ), and HUVECs ( gray bars ); exemplar images shown are of BAECs, and opticin concentration was 250 n m . A , measurements of the total network length formed by BAECs, HRECs, and HUVECs under FGF-2 stimulation in collagen type I showed a significant decrease in presence of opticin (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness *

    doi: 10.1074/jbc.M111.331157

    Figure Lengend Snippet: Opticin inhibits capillary morphogenesis, promotes capillary network regression, and inhibits EC invasion in collagen gels. Three different types of EC were used: BAECs ( black bars ), HRECs ( white bars ), and HUVECs ( gray bars ); exemplar images shown are of BAECs, and opticin concentration was 250 n m . A , measurements of the total network length formed by BAECs, HRECs, and HUVECs under FGF-2 stimulation in collagen type I showed a significant decrease in presence of opticin (*, p

    Article Snippet: Cell Proliferation Assays Using Bromodeoxyuridine (BrdU) Incorporations The effects of recombinant opticin on EC proliferation were tested using BrdU incorporation assays (Calbiochem®, Nottingham, UK).

    Techniques: Concentration Assay

    Rodent Par-4 phosphorylation by CK2 prevents caspase-mediated Par-4 cleavage. ( a ) PC-3 cells transfected with the GFP-tagged constructs wild-type rat Par-4, 124A/D, 223A/D, 124A223A or 124D223D mutants of Par-4 were treated with TRAIL (500 ng/ml, 3 h) in the presence or absence of Z-VAD (15 μ M). Par-4 cleavage was determined by western blotting using anti-GFP antibody. Hsp90 was used as a loading control. ( b ) Wild-type Par-4, 124A/D, 223A/D, 124D223D and 124A223A mutant proteins (GFP tagged) were produced in vitro by a TNT RRL system. Recombinant caspase-8 (upper panel) or caspase-3 and -7 (Casp3, Casp7, lower panel) were incubated with the indicated Par-4 proteins for 3 h at 37 °C, and Par-4 cleavage was assessed by western blotting using GFP antibody. ( c ) Caspase assay was performed on pre-phosphorylated rat GST-Par-4 ( in vitro kinase assay using RecCK2 as described above Figure 2 ), and cleaved Par-4 was detected by immunoblot. *NS: non-specific bands are probably due to the presence of recombinant Par-4 degraded forms. To note: cleaved GST Par-4 migrates at lower molecular weight than cleaved GFP-Par-4, given GST tag is located in the N-terminus of Par-4 and not on C-terminus as the GFP-tag. ( d–f ) PC-3 cells transfected with Par-4 wild-type or FLAG-tagged Par-4 (124–332) were treated as above with TRAIL and analyzed for apoptosis by Hoechst staining ( d ) or FACS analysis of the caspase-3 activity (FLICA) ( e ) and by immunoblotting analysis of caspase-8 and PARP cleavage ( f ). Bars represent the mean±S.D. of at least three independent experiments. Flag-Par-4 (124–332) and GFP were revealed in the same membrane using corresponding antibodies (lower panel). Endogenous Par-4 was used as a loading control. * P

    Journal: Cell Death & Disease

    Article Title: Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells

    doi: 10.1038/cddis.2013.532

    Figure Lengend Snippet: Rodent Par-4 phosphorylation by CK2 prevents caspase-mediated Par-4 cleavage. ( a ) PC-3 cells transfected with the GFP-tagged constructs wild-type rat Par-4, 124A/D, 223A/D, 124A223A or 124D223D mutants of Par-4 were treated with TRAIL (500 ng/ml, 3 h) in the presence or absence of Z-VAD (15 μ M). Par-4 cleavage was determined by western blotting using anti-GFP antibody. Hsp90 was used as a loading control. ( b ) Wild-type Par-4, 124A/D, 223A/D, 124D223D and 124A223A mutant proteins (GFP tagged) were produced in vitro by a TNT RRL system. Recombinant caspase-8 (upper panel) or caspase-3 and -7 (Casp3, Casp7, lower panel) were incubated with the indicated Par-4 proteins for 3 h at 37 °C, and Par-4 cleavage was assessed by western blotting using GFP antibody. ( c ) Caspase assay was performed on pre-phosphorylated rat GST-Par-4 ( in vitro kinase assay using RecCK2 as described above Figure 2 ), and cleaved Par-4 was detected by immunoblot. *NS: non-specific bands are probably due to the presence of recombinant Par-4 degraded forms. To note: cleaved GST Par-4 migrates at lower molecular weight than cleaved GFP-Par-4, given GST tag is located in the N-terminus of Par-4 and not on C-terminus as the GFP-tag. ( d–f ) PC-3 cells transfected with Par-4 wild-type or FLAG-tagged Par-4 (124–332) were treated as above with TRAIL and analyzed for apoptosis by Hoechst staining ( d ) or FACS analysis of the caspase-3 activity (FLICA) ( e ) and by immunoblotting analysis of caspase-8 and PARP cleavage ( f ). Bars represent the mean±S.D. of at least three independent experiments. Flag-Par-4 (124–332) and GFP were revealed in the same membrane using corresponding antibodies (lower panel). Endogenous Par-4 was used as a loading control. * P

    Article Snippet: As a positive control, we used recombinant CK2 (Rec.CK2, 20 000 units), and as a baseline control samples treated with CK2-specific inhibitor tetrabromocinnamic acid (20 μ M, Calbiochem).

    Techniques: Transfection, Construct, Western Blot, Mutagenesis, Produced, In Vitro, Recombinant, Incubation, Caspase Assay, Kinase Assay, Molecular Weight, Staining, FACS, Activity Assay

    Human Par-4 is phosphorylated both in vitro and in vivo on S231 (ortholog of rodent S223) by CK2 and this phosphorylation impairs apoptosis. ( a ) Conservation of the CK2 recognition motif (including the serine 231 of human Par-4) was evaluated in different species. ( b ) Recombinant Myc-tagged human Par-4 (hPar-4) or 231D mutant proteins were produced with TNT RRL system. An in vitro kinase assay was performed with the immunoprecipitates as substrates, in the presence of recombinant CK2 and [ γ −32P]ATP. Phosphorylated Par-4 was detected by autoradiography. Production of recombinant Myc tagged proteins were checked by western blotting (right panel). ( c ) Phosphorylation of endogenous Par-4 was detected by western blotting (left panel), using the human anti-phosphoserine231-Par-4 antibody (Ph231 hPar-4), in PC-3 cells treated (+) or not (−) with TRAIL for 9 h, and densitometry analysis was done. The blocking peptide was used in order to test the specificity of the phospho-antibody. In parallel, the percentage of apoptosis induced by TRAIL (9 h) was assessed by Hoetsch staining. Bars represent the mean±S.D. of at least three independent experiments (right panel). ( d–f ) HCT116 cells were transfected with empty Myc vector control (Mock), human Myc-tagged Par-4, 231A or 231D mutant Par-4 (mimicking unphosphorylated and phosphorylated hPar-4, respectively) and then treated with recombinant TRAIL (150 ng/ml, 3 h). Expression of the different constructs was assessed by western blotting using Myc antibody, and apoptosis was monitored by immunoblot analysis of caspase-8 and PARP cleavage ( d ) and by DAPI staining ( e and f ). Bar graph shows semi-quantified densitometry from PARP and Caspase-8 western blotting analysis. Bars represent the mean±S.D. of at least four independent experiments. Hsp90 was used as a loading control. Bar=10 μ m, magnification × 63; * P

    Journal: Cell Death & Disease

    Article Title: Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells

    doi: 10.1038/cddis.2013.532

    Figure Lengend Snippet: Human Par-4 is phosphorylated both in vitro and in vivo on S231 (ortholog of rodent S223) by CK2 and this phosphorylation impairs apoptosis. ( a ) Conservation of the CK2 recognition motif (including the serine 231 of human Par-4) was evaluated in different species. ( b ) Recombinant Myc-tagged human Par-4 (hPar-4) or 231D mutant proteins were produced with TNT RRL system. An in vitro kinase assay was performed with the immunoprecipitates as substrates, in the presence of recombinant CK2 and [ γ −32P]ATP. Phosphorylated Par-4 was detected by autoradiography. Production of recombinant Myc tagged proteins were checked by western blotting (right panel). ( c ) Phosphorylation of endogenous Par-4 was detected by western blotting (left panel), using the human anti-phosphoserine231-Par-4 antibody (Ph231 hPar-4), in PC-3 cells treated (+) or not (−) with TRAIL for 9 h, and densitometry analysis was done. The blocking peptide was used in order to test the specificity of the phospho-antibody. In parallel, the percentage of apoptosis induced by TRAIL (9 h) was assessed by Hoetsch staining. Bars represent the mean±S.D. of at least three independent experiments (right panel). ( d–f ) HCT116 cells were transfected with empty Myc vector control (Mock), human Myc-tagged Par-4, 231A or 231D mutant Par-4 (mimicking unphosphorylated and phosphorylated hPar-4, respectively) and then treated with recombinant TRAIL (150 ng/ml, 3 h). Expression of the different constructs was assessed by western blotting using Myc antibody, and apoptosis was monitored by immunoblot analysis of caspase-8 and PARP cleavage ( d ) and by DAPI staining ( e and f ). Bar graph shows semi-quantified densitometry from PARP and Caspase-8 western blotting analysis. Bars represent the mean±S.D. of at least four independent experiments. Hsp90 was used as a loading control. Bar=10 μ m, magnification × 63; * P

    Article Snippet: As a positive control, we used recombinant CK2 (Rec.CK2, 20 000 units), and as a baseline control samples treated with CK2-specific inhibitor tetrabromocinnamic acid (20 μ M, Calbiochem).

    Techniques: In Vitro, In Vivo, Recombinant, Mutagenesis, Produced, Kinase Assay, Autoradiography, Western Blot, Blocking Assay, Staining, Transfection, Plasmid Preparation, Expressing, Construct

    Identification of the CK2 phosphorylated sites S124 and S223 in rodent Par-4. ( a ) Two-dimensional phosphopeptide map analysis of the phosphorylated recombinant rat GST-Par-4. GST-Par-4 was phosphorylated by recombinant CK2 in the presence of [ γ −32P]ATP. After migration, band corresponding to the radiolabeled phospho-Par-4 was excised from the membrane and digested by trypsin. Tryptic fragments were purified and resolved by two-dimensional thin layer electrophoresis and ascending chromatography followed by autoradiography to visualize the phosphopeptides. The separation origin is indicated as ‘0'. Three major phosphopeptides were revealed by autoradiography: P1, P1′, and P2. ( b ) Transfected wild-type Par-4 (rat) or 124A223A Par-4 mutants (GFP-tagged) were immunoprecipitated from COS cells and subjected to an in vitro kinase assay in the presence of recombinant CK2 and [γ−32P]ATP. Radiolabeled Par-4 was visualized upon autoradiography. Immunoblot analysis confirmed that equal amounts of GFP-Par-4 and GFP-124A223A were immunoprecipitated from each cell lysate (inputs, lower panel)

    Journal: Cell Death & Disease

    Article Title: Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells

    doi: 10.1038/cddis.2013.532

    Figure Lengend Snippet: Identification of the CK2 phosphorylated sites S124 and S223 in rodent Par-4. ( a ) Two-dimensional phosphopeptide map analysis of the phosphorylated recombinant rat GST-Par-4. GST-Par-4 was phosphorylated by recombinant CK2 in the presence of [ γ −32P]ATP. After migration, band corresponding to the radiolabeled phospho-Par-4 was excised from the membrane and digested by trypsin. Tryptic fragments were purified and resolved by two-dimensional thin layer electrophoresis and ascending chromatography followed by autoradiography to visualize the phosphopeptides. The separation origin is indicated as ‘0'. Three major phosphopeptides were revealed by autoradiography: P1, P1′, and P2. ( b ) Transfected wild-type Par-4 (rat) or 124A223A Par-4 mutants (GFP-tagged) were immunoprecipitated from COS cells and subjected to an in vitro kinase assay in the presence of recombinant CK2 and [γ−32P]ATP. Radiolabeled Par-4 was visualized upon autoradiography. Immunoblot analysis confirmed that equal amounts of GFP-Par-4 and GFP-124A223A were immunoprecipitated from each cell lysate (inputs, lower panel)

    Article Snippet: As a positive control, we used recombinant CK2 (Rec.CK2, 20 000 units), and as a baseline control samples treated with CK2-specific inhibitor tetrabromocinnamic acid (20 μ M, Calbiochem).

    Techniques: Recombinant, Migration, Purification, Electrophoresis, Chromatography, Autoradiography, Transfection, Immunoprecipitation, In Vitro, Kinase Assay

    Anti-apoptotic role of CK2 is dependent on Par-4 in human prostate cancer cells. ( a–d ) PC-3 cells were transfected with different CK2 siRNAs (siCK2T from ThermoFischer ( a ) or siCK2AB from Ambion ( b )) together with scrambled siRNA fluorescently labeled with FAM (Scr siRNA) or different Par-4 siRNA (siPar-4T from ThermoFisher or siPar-4sc from Santa Cruz) for 48 h. Then, cells were treated or not with TRAIL (500 ng/ml, 3 h). The downregulation of Par-4 and CK2 α proteins was confirmed by immunoblot using the corresponding antibodies. Apoptosis was monitored ( a and b ) by immunoblot analysis of caspase-8 and PARP (long and short cleaved PARP forms) cleavage and ( c and d ) by DAPI staining. Graphs represent semi-quantified densitometry analysis from PARP and caspase-8 western blotting analysis of panel ( a ). See also Supplementary Figure S10A for the densitometric analysis of panel ( b ). Bars represent the mean±S.D. of at least four independent experiments. Hsp90 was used as a loading control. Bar=10 μ m, magnification × 63; * P

    Journal: Cell Death & Disease

    Article Title: Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells

    doi: 10.1038/cddis.2013.532

    Figure Lengend Snippet: Anti-apoptotic role of CK2 is dependent on Par-4 in human prostate cancer cells. ( a–d ) PC-3 cells were transfected with different CK2 siRNAs (siCK2T from ThermoFischer ( a ) or siCK2AB from Ambion ( b )) together with scrambled siRNA fluorescently labeled with FAM (Scr siRNA) or different Par-4 siRNA (siPar-4T from ThermoFisher or siPar-4sc from Santa Cruz) for 48 h. Then, cells were treated or not with TRAIL (500 ng/ml, 3 h). The downregulation of Par-4 and CK2 α proteins was confirmed by immunoblot using the corresponding antibodies. Apoptosis was monitored ( a and b ) by immunoblot analysis of caspase-8 and PARP (long and short cleaved PARP forms) cleavage and ( c and d ) by DAPI staining. Graphs represent semi-quantified densitometry analysis from PARP and caspase-8 western blotting analysis of panel ( a ). See also Supplementary Figure S10A for the densitometric analysis of panel ( b ). Bars represent the mean±S.D. of at least four independent experiments. Hsp90 was used as a loading control. Bar=10 μ m, magnification × 63; * P

    Article Snippet: As a positive control, we used recombinant CK2 (Rec.CK2, 20 000 units), and as a baseline control samples treated with CK2-specific inhibitor tetrabromocinnamic acid (20 μ M, Calbiochem).

    Techniques: Transfection, Labeling, Staining, Western Blot

    Phosphorylation of rodent Par-4 by CK2 blocks Par-4 proapoptotic functions. ( a–c ) PC-3 cells were transfected with empty GFP vector control (Mock), rat GFP-tagged Par-4, GFP-tagged 124A223A mutant (mimicking non-phosphorylated Par-4 form) or 124D223D mutant (mimicking phosphorylated Par-4 form) and then treated with recombinant death ligand TRAIL (500 ng/ml, 3 h). ( a ) Green fluorescent (transfected) cells were gated (represent around 20% of total cell) and further analyzed for apoptosis by Hoechst staining. Bars represent the mean±S.D. of at least four independent experiments. ( b ) FACS analysis of the caspase-3 activity was assessed by red fluorescent signal FLICA (Fluorogenic inhibitors of caspase-3 activation). Bars represent the mean±S.D. of at least four independent experiments. ( c ) Immunoblot analysis of caspase-8 and PARP cleavage (included both GFP-positive and -negative cells) at the indicated times. * P

    Journal: Cell Death & Disease

    Article Title: Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells

    doi: 10.1038/cddis.2013.532

    Figure Lengend Snippet: Phosphorylation of rodent Par-4 by CK2 blocks Par-4 proapoptotic functions. ( a–c ) PC-3 cells were transfected with empty GFP vector control (Mock), rat GFP-tagged Par-4, GFP-tagged 124A223A mutant (mimicking non-phosphorylated Par-4 form) or 124D223D mutant (mimicking phosphorylated Par-4 form) and then treated with recombinant death ligand TRAIL (500 ng/ml, 3 h). ( a ) Green fluorescent (transfected) cells were gated (represent around 20% of total cell) and further analyzed for apoptosis by Hoechst staining. Bars represent the mean±S.D. of at least four independent experiments. ( b ) FACS analysis of the caspase-3 activity was assessed by red fluorescent signal FLICA (Fluorogenic inhibitors of caspase-3 activation). Bars represent the mean±S.D. of at least four independent experiments. ( c ) Immunoblot analysis of caspase-8 and PARP cleavage (included both GFP-positive and -negative cells) at the indicated times. * P

    Article Snippet: As a positive control, we used recombinant CK2 (Rec.CK2, 20 000 units), and as a baseline control samples treated with CK2-specific inhibitor tetrabromocinnamic acid (20 μ M, Calbiochem).

    Techniques: Transfection, Plasmid Preparation, Mutagenesis, Recombinant, Staining, FACS, Activity Assay, Activation Assay

    Both rodent and human Par-4 interact and are substrates of CK2. ( a ) COS cells were co-transfected with rat GFP-tagged Par-4 or empty GFP vector together with the CK2 α and/or the CK2 β HA-tagged subunits. In the left panel, immunoprecipitation of GFP vector, Par-4 (GFP) or CK2 α subunits (HA). In right panel, immunoprecipitation of Par-4 (GFP) or CK2 α and/or CK2 β subunits (HA). The immunoprecipitations were followed by immunodetection of either CK2 (HA) or Par-4 (GFP). ( b ) Immunoprecipitation, from a same PC-3 cells extract, of endogenous human Par-4 (hPar-4) or CK2 subunits was followed by immunodetection of endogenous human CK2 α , CK2 β or Par-4. Inputs: proteins in total cell lysates. IP IgG: immunoprecipitation with a non-relevant antibody (IgG mouse). *HC, LC immunoglobulin heavy chain and light chain respectively. ( c ) The GFP-tagged CK2 kinase or GFP alone (as a control, Mock) were immunoprecipitated from transfected COS cells and used for an i n vitro kinase assay in the presence of [ γ −32P]ATP with either recombinant GST-Par-4 or GST alone, as substrates (left panel). Reaction products were resolved by SDS-PAGE on 10% gels, stained with Coomassie blue to verify that equal amounts of GST-Par-4 or GST alone were used in each reaction (lower panel), and autoradiography was performed (upper panel). In parallel, the amount of GFP (Mock) or CK2 subunits immunoprecipitated was analyzed by immunoblotting (right panel). ( d ) Recombinant GFP-tagged human Par-4 (GFP-hPar-4) was produced in vitro by the TNT rabbit reticulocyte lysate system (RRL), and lysates were subsequently immunoprecipitated using GFP antibody. Used as substrates, the immunoprecipitates were subjected to an in vitro kinase assay using recombinant CK2 (recCK2) in the presence of [ γ −32P]ATP for 30 min. The CK2 inhibitor (TBB, 1 μ M) was incubated 5 min before addition of CK2 recombinant (middle panel). After migration, phosphorylated Par-4 ( 32 P-hPar-4) and autophosphorylation of recombinant CK2 β ( 32 P- CK2 β ) was detected by autoradiography. The amount of recombinant GFP-Par-4 produced by RRL was checked by western blotting (input, lower panel)

    Journal: Cell Death & Disease

    Article Title: Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells

    doi: 10.1038/cddis.2013.532

    Figure Lengend Snippet: Both rodent and human Par-4 interact and are substrates of CK2. ( a ) COS cells were co-transfected with rat GFP-tagged Par-4 or empty GFP vector together with the CK2 α and/or the CK2 β HA-tagged subunits. In the left panel, immunoprecipitation of GFP vector, Par-4 (GFP) or CK2 α subunits (HA). In right panel, immunoprecipitation of Par-4 (GFP) or CK2 α and/or CK2 β subunits (HA). The immunoprecipitations were followed by immunodetection of either CK2 (HA) or Par-4 (GFP). ( b ) Immunoprecipitation, from a same PC-3 cells extract, of endogenous human Par-4 (hPar-4) or CK2 subunits was followed by immunodetection of endogenous human CK2 α , CK2 β or Par-4. Inputs: proteins in total cell lysates. IP IgG: immunoprecipitation with a non-relevant antibody (IgG mouse). *HC, LC immunoglobulin heavy chain and light chain respectively. ( c ) The GFP-tagged CK2 kinase or GFP alone (as a control, Mock) were immunoprecipitated from transfected COS cells and used for an i n vitro kinase assay in the presence of [ γ −32P]ATP with either recombinant GST-Par-4 or GST alone, as substrates (left panel). Reaction products were resolved by SDS-PAGE on 10% gels, stained with Coomassie blue to verify that equal amounts of GST-Par-4 or GST alone were used in each reaction (lower panel), and autoradiography was performed (upper panel). In parallel, the amount of GFP (Mock) or CK2 subunits immunoprecipitated was analyzed by immunoblotting (right panel). ( d ) Recombinant GFP-tagged human Par-4 (GFP-hPar-4) was produced in vitro by the TNT rabbit reticulocyte lysate system (RRL), and lysates were subsequently immunoprecipitated using GFP antibody. Used as substrates, the immunoprecipitates were subjected to an in vitro kinase assay using recombinant CK2 (recCK2) in the presence of [ γ −32P]ATP for 30 min. The CK2 inhibitor (TBB, 1 μ M) was incubated 5 min before addition of CK2 recombinant (middle panel). After migration, phosphorylated Par-4 ( 32 P-hPar-4) and autophosphorylation of recombinant CK2 β ( 32 P- CK2 β ) was detected by autoradiography. The amount of recombinant GFP-Par-4 produced by RRL was checked by western blotting (input, lower panel)

    Article Snippet: As a positive control, we used recombinant CK2 (Rec.CK2, 20 000 units), and as a baseline control samples treated with CK2-specific inhibitor tetrabromocinnamic acid (20 μ M, Calbiochem).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Immunodetection, Kinase Assay, Recombinant, SDS Page, Staining, Autoradiography, Produced, In Vitro, Incubation, Migration, Western Blot

    Par-4 is highly phosphorylated on S231 (ortholog of rodent S223) in human prostate cancer cells compared with normal counterparts. ( a ) Upper panel, phosphorylation of Par-4 on S231 was studied by western blotting in prostate cancer cells (PC-3, LnCap cells) and in normal prostate cells (PrCE, PNT2C2). In parallel, endogenous expression of Par-4 and CK2 subunits in the different cell lines was determined (middle panel). Hsp90 was used as a loading control. Lower panel, analysis of CK2 activity in the different prostate cells studied using Cyclex CK2 screening kit. Bars represent the mean±S.D. of at least two independent experiments. ( b ) PC-3 cells were transfected with CK2 siRNA (siCK2T, ThermoFischer) or scrambled siRNA fluorescently labeled with FAM (Scr siRNA) for 48 h and then treated or not with TRAIL (500 ng/ml, 3 h). Endogenous phosphorylated human Par-4 was detected by western blotting using the anti-phospho231 Par-4 antibody (Ph231-hPar-4) (left panel). Par-4 expression (left panel) and downregulation of CK2 α protein (right panel) were evaluated by immunoblotting using the corresponding antibodies. Bar graph shows semi-quantified densitometry from western blotting analysis

    Journal: Cell Death & Disease

    Article Title: Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells

    doi: 10.1038/cddis.2013.532

    Figure Lengend Snippet: Par-4 is highly phosphorylated on S231 (ortholog of rodent S223) in human prostate cancer cells compared with normal counterparts. ( a ) Upper panel, phosphorylation of Par-4 on S231 was studied by western blotting in prostate cancer cells (PC-3, LnCap cells) and in normal prostate cells (PrCE, PNT2C2). In parallel, endogenous expression of Par-4 and CK2 subunits in the different cell lines was determined (middle panel). Hsp90 was used as a loading control. Lower panel, analysis of CK2 activity in the different prostate cells studied using Cyclex CK2 screening kit. Bars represent the mean±S.D. of at least two independent experiments. ( b ) PC-3 cells were transfected with CK2 siRNA (siCK2T, ThermoFischer) or scrambled siRNA fluorescently labeled with FAM (Scr siRNA) for 48 h and then treated or not with TRAIL (500 ng/ml, 3 h). Endogenous phosphorylated human Par-4 was detected by western blotting using the anti-phospho231 Par-4 antibody (Ph231-hPar-4) (left panel). Par-4 expression (left panel) and downregulation of CK2 α protein (right panel) were evaluated by immunoblotting using the corresponding antibodies. Bar graph shows semi-quantified densitometry from western blotting analysis

    Article Snippet: As a positive control, we used recombinant CK2 (Rec.CK2, 20 000 units), and as a baseline control samples treated with CK2-specific inhibitor tetrabromocinnamic acid (20 μ M, Calbiochem).

    Techniques: Western Blot, Expressing, Activity Assay, Transfection, Labeling

    Generation of MyD88-5 knockout mice and protection of their hippocampal neurons from OGD-induced cell death. (A) Schematic of the MyD88-5 locus, targeting vector, and predicted structure of the mutated locus. Exon numbers are indicated. The probe used for Southern blotting is shown in red. Restriction enzyme sites and predicted sizes of the digests are shown. Sp, SphI; Neo, neomycin resistance gene; DTX, diphtheria toxin gene. (B) Southern blot analysis of genomic DNA from MyD88-5 wild-type, hemizygous-deficient, and null mice. (C) Western blot analysis of MyD88-5 expression in wild-type, hemizygous-deficient, and null mice. 50 μg of brain lysates were subjected to SDS-PAGE. Affinity-purified chicken anti–MyD88-5 antibody was used for detection. (D) Propidium iodide staining of dead neurons in hippocampal slices at the start of the OGD period (basal), after OGD (OGD), or the same period without OGD treatment (sham), and after exposure to 1 mM NMDA to induce a maximal cell death (Max). Hippocampi illustrated were from MyD88-5 +/− (Het) or MyD88-5 −/− mice (KO). Bar, 1 mm. (E) Quantitative evaluation of cell death after sham or OGD treatment in MyD88-5 +/+ (sham, n = 6; OGD, n = 13) MyD88-5 +/− (sham, n = 11; OGD, n = 22) and MyD88-5 −/− (sham, n = 5; OGD, n = 29) slices. P

    Journal: The Journal of Experimental Medicine

    Article Title: MyD88-5 links mitochondria, microtubules, and JNK3 in neurons and regulates neuronal survival

    doi: 10.1084/jem.20070868

    Figure Lengend Snippet: Generation of MyD88-5 knockout mice and protection of their hippocampal neurons from OGD-induced cell death. (A) Schematic of the MyD88-5 locus, targeting vector, and predicted structure of the mutated locus. Exon numbers are indicated. The probe used for Southern blotting is shown in red. Restriction enzyme sites and predicted sizes of the digests are shown. Sp, SphI; Neo, neomycin resistance gene; DTX, diphtheria toxin gene. (B) Southern blot analysis of genomic DNA from MyD88-5 wild-type, hemizygous-deficient, and null mice. (C) Western blot analysis of MyD88-5 expression in wild-type, hemizygous-deficient, and null mice. 50 μg of brain lysates were subjected to SDS-PAGE. Affinity-purified chicken anti–MyD88-5 antibody was used for detection. (D) Propidium iodide staining of dead neurons in hippocampal slices at the start of the OGD period (basal), after OGD (OGD), or the same period without OGD treatment (sham), and after exposure to 1 mM NMDA to induce a maximal cell death (Max). Hippocampi illustrated were from MyD88-5 +/− (Het) or MyD88-5 −/− mice (KO). Bar, 1 mm. (E) Quantitative evaluation of cell death after sham or OGD treatment in MyD88-5 +/+ (sham, n = 6; OGD, n = 13) MyD88-5 +/− (sham, n = 11; OGD, n = 22) and MyD88-5 −/− (sham, n = 5; OGD, n = 29) slices. P

    Article Snippet: Recombinant MyD88-5 was concentrated with a Centricon column (10 kD cut-off; Millipore) and used for immunization of chickens to generate anti–mouse MyD88-5 fowl immunoglobulin (IgY).

    Techniques: Knock-Out, Mouse Assay, Plasmid Preparation, Southern Blot, Western Blot, Expressing, SDS Page, Affinity Purification, Staining

    MyD88-5–dependent recruitment of JNK3 to mitochondria. (A) Redistribution of JNK3 to mitochondria in the presence of MyD88-5. COS-1 cells were transfected with MyD88-5/GFP and RFP (top), GFP and JNK3/RFP (middle), or MyD88-5/GFP and JNK3/RFP (bottom). Mitochondria were stained with MitoTracker Red CMXRos. Bar, 10 μm. (B) Coimmunoprecipitation of MyD88-5 with JNK3/GFP, but not with GFP alone, from transfected COS-1 cells. Immunoprecipitation was with anti-GFP antibody and protein G–Sepharose and Western blotting was with antibody to MyD88-5 (top) or GFP (bottom). Open arrowhead indicates nonspecific bands. (C) Coimmunoprecipitation of MyD88-5 with JNK3 in hippocampal slices from wild-type (WT) or MyD88-5 knockout (KO) mice. Half of the brain slices were preexposed to 120 Joules/m 2 of UV light. Immunoprecipitation was done with anti-JNK1,3 and protein G–Sepharose and Western blot with anti-JNK3 and anti-MyD88-5.

    Journal: The Journal of Experimental Medicine

    Article Title: MyD88-5 links mitochondria, microtubules, and JNK3 in neurons and regulates neuronal survival

    doi: 10.1084/jem.20070868

    Figure Lengend Snippet: MyD88-5–dependent recruitment of JNK3 to mitochondria. (A) Redistribution of JNK3 to mitochondria in the presence of MyD88-5. COS-1 cells were transfected with MyD88-5/GFP and RFP (top), GFP and JNK3/RFP (middle), or MyD88-5/GFP and JNK3/RFP (bottom). Mitochondria were stained with MitoTracker Red CMXRos. Bar, 10 μm. (B) Coimmunoprecipitation of MyD88-5 with JNK3/GFP, but not with GFP alone, from transfected COS-1 cells. Immunoprecipitation was with anti-GFP antibody and protein G–Sepharose and Western blotting was with antibody to MyD88-5 (top) or GFP (bottom). Open arrowhead indicates nonspecific bands. (C) Coimmunoprecipitation of MyD88-5 with JNK3 in hippocampal slices from wild-type (WT) or MyD88-5 knockout (KO) mice. Half of the brain slices were preexposed to 120 Joules/m 2 of UV light. Immunoprecipitation was done with anti-JNK1,3 and protein G–Sepharose and Western blot with anti-JNK3 and anti-MyD88-5.

    Article Snippet: Recombinant MyD88-5 was concentrated with a Centricon column (10 kD cut-off; Millipore) and used for immunization of chickens to generate anti–mouse MyD88-5 fowl immunoglobulin (IgY).

    Techniques: Transfection, Staining, Immunoprecipitation, Western Blot, Knock-Out, Mouse Assay

    Mitochondrial association of MyD88-5. (A) Selective colocalization of MyD88-5/GFP fusion protein with mitochondria and microtubules. COS-1 cells were transfected with a MyD88-5/GFP fusion construct. 18 h after the transfection, cells were stained with MitoTracker Red CMXRos for 20 min and fixed, or fixed, permeabilized, and stained with antibody for Golgi (anti–γ-adaptin) or microtubules (anti-tubulin). (B) Extrinsic association of MyD88-5 with mitochondria. Confocal microscopic images of COS-1 cells transfected with MyD88-5/GFP and stained with MitoTracker Red CMXRos. (top) Cells expressing lower levels of MyD88-5; (bottom) cells expressing higher levels of MyD88-5, with effects on mitochondrial shape and localization. (right) Magnified images of the boxed areas from the images on the left. (C) Analysis of domains of MyD88-5 contributing to subcellular localization and mitochondrial association. COS-1 cells were transfected with the indicated constructs. 18 h later, mitochondria were stained with MitoTracker Red CMXRos before fixation. (D) Partial copurification of endogenous MyD88-5 and transgenic MyD88-5/GFP with mitochondria from brain. Mitochondria were isolated from wild type (non-Tg) or transgenic (Tg) mouse brains by differential centrifugation and lysates were Western blotted with antibody to MyD88-5 (top), GFP (middle), or TOM-23 (bottom). TOM-23 is an intrinsic mitochondrial membrane protein whose relative abundance serves as a loading control. (E) Copurification of endogenous MyD88-5 and microtubules from mouse brain. Microtubules were either polymerized with Taxol and GTP at 37°C or prevented from polymerization with colchicine at 4°C before purification by sucrose gradient centrifugation. Purified microtubule fractions (pellet), unpolymerized fractions (sup), and total brain lysate (total lysate) were Western blotted with antibody to MyD88-5. Bars, 10 μm.

    Journal: The Journal of Experimental Medicine

    Article Title: MyD88-5 links mitochondria, microtubules, and JNK3 in neurons and regulates neuronal survival

    doi: 10.1084/jem.20070868

    Figure Lengend Snippet: Mitochondrial association of MyD88-5. (A) Selective colocalization of MyD88-5/GFP fusion protein with mitochondria and microtubules. COS-1 cells were transfected with a MyD88-5/GFP fusion construct. 18 h after the transfection, cells were stained with MitoTracker Red CMXRos for 20 min and fixed, or fixed, permeabilized, and stained with antibody for Golgi (anti–γ-adaptin) or microtubules (anti-tubulin). (B) Extrinsic association of MyD88-5 with mitochondria. Confocal microscopic images of COS-1 cells transfected with MyD88-5/GFP and stained with MitoTracker Red CMXRos. (top) Cells expressing lower levels of MyD88-5; (bottom) cells expressing higher levels of MyD88-5, with effects on mitochondrial shape and localization. (right) Magnified images of the boxed areas from the images on the left. (C) Analysis of domains of MyD88-5 contributing to subcellular localization and mitochondrial association. COS-1 cells were transfected with the indicated constructs. 18 h later, mitochondria were stained with MitoTracker Red CMXRos before fixation. (D) Partial copurification of endogenous MyD88-5 and transgenic MyD88-5/GFP with mitochondria from brain. Mitochondria were isolated from wild type (non-Tg) or transgenic (Tg) mouse brains by differential centrifugation and lysates were Western blotted with antibody to MyD88-5 (top), GFP (middle), or TOM-23 (bottom). TOM-23 is an intrinsic mitochondrial membrane protein whose relative abundance serves as a loading control. (E) Copurification of endogenous MyD88-5 and microtubules from mouse brain. Microtubules were either polymerized with Taxol and GTP at 37°C or prevented from polymerization with colchicine at 4°C before purification by sucrose gradient centrifugation. Purified microtubule fractions (pellet), unpolymerized fractions (sup), and total brain lysate (total lysate) were Western blotted with antibody to MyD88-5. Bars, 10 μm.

    Article Snippet: Recombinant MyD88-5 was concentrated with a Centricon column (10 kD cut-off; Millipore) and used for immunization of chickens to generate anti–mouse MyD88-5 fowl immunoglobulin (IgY).

    Techniques: Transfection, Construct, Staining, Expressing, Copurification, Transgenic Assay, Isolation, Centrifugation, Western Blot, Purification, Gradient Centrifugation

    MyD88-5 is expressed mainly in brain, but not in myeloid cells. (A) Northern blot analysis of MyD88-5 expression in mouse tissues using the two SAM domains and part of the TIR domain (1,200–1,816 bp) as a probe. An actin probe was used as a loading control. (B) Western blot with chicken antibody raised against full-length recombinant mouse MyD88-5 reveals a 79-kD polypeptide in mouse brain (left lane) and in COS-1 cells transfected with a Flag-tagged MyD88-5 construct (right lane, filled arrowhead), but not in vector-transfected COS-1 cells (center lane). The open arrowhead indicates a nonspecific band. (C) Relative quantitation of MyD88-5 transcripts in mouse tissues and cells. Expression was highest in brain and moderate in lymph node (LN) and purified splenic T cells, but was barely detectable in splenic populations (chiefly macrophages) depleted of T and B cells (T- B-). Data are quantitative real-time PCR results normalized to mouse GAPDH and expressed as the percentage of the level in brain. One of three independent experiments is shown. (D) Relative quantitation of MyD88-5 transcripts in human tissues and cells. Expression of human MyD88-5 mRNA is highest in brain and detectable in lymphocytes (L). Polymorphonuclear leukocytes (PMN), monocytes (M), and lymphocytes were purified from human blood. Data are expressed as in C for one of three independent experiments. (E) MyD88-5 expression in various anatomic regions of adult mouse brain. Data are expressed as in C, and are from one of two independent experiments. (F) MyD88-5 expression in mouse brain during development. E12-18, embryonic days after conception; P1-60, postnatal days. qRT-PCR results are normalized to GAPDH and shown as the percentage of MyD88-5 expression in adult brain.

    Journal: The Journal of Experimental Medicine

    Article Title: MyD88-5 links mitochondria, microtubules, and JNK3 in neurons and regulates neuronal survival

    doi: 10.1084/jem.20070868

    Figure Lengend Snippet: MyD88-5 is expressed mainly in brain, but not in myeloid cells. (A) Northern blot analysis of MyD88-5 expression in mouse tissues using the two SAM domains and part of the TIR domain (1,200–1,816 bp) as a probe. An actin probe was used as a loading control. (B) Western blot with chicken antibody raised against full-length recombinant mouse MyD88-5 reveals a 79-kD polypeptide in mouse brain (left lane) and in COS-1 cells transfected with a Flag-tagged MyD88-5 construct (right lane, filled arrowhead), but not in vector-transfected COS-1 cells (center lane). The open arrowhead indicates a nonspecific band. (C) Relative quantitation of MyD88-5 transcripts in mouse tissues and cells. Expression was highest in brain and moderate in lymph node (LN) and purified splenic T cells, but was barely detectable in splenic populations (chiefly macrophages) depleted of T and B cells (T- B-). Data are quantitative real-time PCR results normalized to mouse GAPDH and expressed as the percentage of the level in brain. One of three independent experiments is shown. (D) Relative quantitation of MyD88-5 transcripts in human tissues and cells. Expression of human MyD88-5 mRNA is highest in brain and detectable in lymphocytes (L). Polymorphonuclear leukocytes (PMN), monocytes (M), and lymphocytes were purified from human blood. Data are expressed as in C for one of three independent experiments. (E) MyD88-5 expression in various anatomic regions of adult mouse brain. Data are expressed as in C, and are from one of two independent experiments. (F) MyD88-5 expression in mouse brain during development. E12-18, embryonic days after conception; P1-60, postnatal days. qRT-PCR results are normalized to GAPDH and shown as the percentage of MyD88-5 expression in adult brain.

    Article Snippet: Recombinant MyD88-5 was concentrated with a Centricon column (10 kD cut-off; Millipore) and used for immunization of chickens to generate anti–mouse MyD88-5 fowl immunoglobulin (IgY).

    Techniques: Northern Blot, Expressing, Western Blot, Recombinant, Transfection, Construct, Plasmid Preparation, Quantitation Assay, Purification, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Generation of transgenic mice expressing MyD88-5-GFP fusion protein and its expression in neurons in the brain. (A) Schematic of the MyD88-5 locus in BAC clone RP23-399H5, the targeting vector, and the predicted structure of the modified MyD88-5 locus. Exon numbers are indicated. Probe used for Southern blotting is shown in red. Restriction sites and predicted fragment sizes are indicated. The stop codon in exon 9 is removed, and GFP is introduced in-frame. (B) Representative Southern blot of restricted genomic DNA from transgenic (Tg) and nontransgenic mice. Transgene copy numbers shown below are calculated by comparing the intensity of the endogenous band (filled arrowhead) and transgenic band (open arrowhead). (C) Expression of endogenous MyD88-5 and MyD88-5/GFP fusion protein in transgenic mouse brain. (left) Western blot with anti–MyD88-5 antibody. (right) Western blot with anti-GFP antibody. The fusion protein has the expected molecular weight of 105 kD. (D) Widespread expression of MyD88-5/GFP in the brain of transgenic mice. Brain sections from a nontransgenic mouse (left) and a transgenic (right) mouse were stained together with anti-GFP antibody. (E) Neuronal expression of MyD88-5/GFP. Fluorescent confocal microscopy captured direct GFP signals from unstained brain sections from a transgenic founder mouse. Right images are magnifications of the insets from the left. Bars: (D) 1 mm; (E, left) 200 μm; (E, right) 20 μm.

    Journal: The Journal of Experimental Medicine

    Article Title: MyD88-5 links mitochondria, microtubules, and JNK3 in neurons and regulates neuronal survival

    doi: 10.1084/jem.20070868

    Figure Lengend Snippet: Generation of transgenic mice expressing MyD88-5-GFP fusion protein and its expression in neurons in the brain. (A) Schematic of the MyD88-5 locus in BAC clone RP23-399H5, the targeting vector, and the predicted structure of the modified MyD88-5 locus. Exon numbers are indicated. Probe used for Southern blotting is shown in red. Restriction sites and predicted fragment sizes are indicated. The stop codon in exon 9 is removed, and GFP is introduced in-frame. (B) Representative Southern blot of restricted genomic DNA from transgenic (Tg) and nontransgenic mice. Transgene copy numbers shown below are calculated by comparing the intensity of the endogenous band (filled arrowhead) and transgenic band (open arrowhead). (C) Expression of endogenous MyD88-5 and MyD88-5/GFP fusion protein in transgenic mouse brain. (left) Western blot with anti–MyD88-5 antibody. (right) Western blot with anti-GFP antibody. The fusion protein has the expected molecular weight of 105 kD. (D) Widespread expression of MyD88-5/GFP in the brain of transgenic mice. Brain sections from a nontransgenic mouse (left) and a transgenic (right) mouse were stained together with anti-GFP antibody. (E) Neuronal expression of MyD88-5/GFP. Fluorescent confocal microscopy captured direct GFP signals from unstained brain sections from a transgenic founder mouse. Right images are magnifications of the insets from the left. Bars: (D) 1 mm; (E, left) 200 μm; (E, right) 20 μm.

    Article Snippet: Recombinant MyD88-5 was concentrated with a Centricon column (10 kD cut-off; Millipore) and used for immunization of chickens to generate anti–mouse MyD88-5 fowl immunoglobulin (IgY).

    Techniques: Transgenic Assay, Mouse Assay, Expressing, BAC Assay, Plasmid Preparation, Modification, Southern Blot, Western Blot, Molecular Weight, Staining, Confocal Microscopy