chromatographic column  (Millipore)


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    Aldrich chromatography column
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    z547875
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    Millipore chromatographic column
    Aldrich chromatography column

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    Article Title: The Immunological Properties of Stroma-free Polyhemolysate Containing Catalase and Superoxide Dismutase Activities Prepared by Polymerized Bovine Stroma-free Hemolysate
    Article Snippet: .. The following materials were purchased from different sources as shown: fresh bovine blood (McGill University, MacDonald campus), NaCl (Fisher), Potassium Phosphate (Sigma), Toluene (Sigma), Sodium Phosphate (Sigma), 99% Lysine Monohydrochloride (Sigma), 25% Glutaraldehyde (Sigma), 1.5cm*98cm Chromatography Column (Fisher), Sepacryl S-300HR (Sigma), Tris Base (Sigma), 37% Hydrochloric Acid (Sigma), Catalase from bovine liver (Sigma), Superoxide Dismuatase from bovine erythrocyte (Sigma), 30% Brij 35 solution (Sigma), Drabkin (Sigma), Molecular Weight Marker Kit (29,000–700,000) (Sigma), UltraPure Water (Cayman Chemical), 100kDa Centrifugal Filter (Millipore), 14.1 g/dl Haemiglobincyanide Standard (J.T. .. Baker), Catalase Assay Kit (Sigma), Superoxide Dismutase Kit (R & D Systems), 3%(w/w) Hydrogen Peroxide (Sigma), Sodium Perborate Tetrahydrate (Sigma), 95–98% Sulfuric Acid (Sigma), 99% Potassium Permanganate (Sigma), Hepalean 1000U.S.P. units/ml (Organon), Pentobarbital Sodique (CEVA Sante Animal), Complement C3a des Arg ELISA Kits (Assay Design), Sodium Hydroxide (Sigma), 99.9% Sodium Azide (Sigma), Agar (Sigma).

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    Article Title: Comparative Analysis of Human γD-Crystallin Aggregation under Physiological and Low pH Conditions
    Article Snippet: .. Materials Salts, tryptone, yeast extract, and chromatography columns were purchased from Sigma (USA). .. Kanamycin, imidazole, and isopropyl β-D-thiolgalactorpyranoside (IPTG) were obtained from Biobasic (Canada).

    Article Title: 3D Imaging of Axons in Transparent Spinal Cords from Rodents and Nonhuman Primates Imaging of Axons in Transparent Spinal Cords from Rodents and Nonhuman Primates Imaging of Axons in Transparent Spinal Cords from Rodents and Nonhuman Primates 1
    Article Snippet: .. Peroxides were removed from THF by passing 100% THF through a chromatography column filled with basic activated aluminum oxide (Sigma 199443) as previously described ( ). (Warning: this process also removes the stabilizer from THF, which may explode after prolonged exposure to oxygen or sunlight. .. Thus, users must add 250 mg/l butylated hydroxytoluene (Sigma W218405) to THF after peroxide removal.)

    Article Title: The Immunological Properties of Stroma-free Polyhemolysate Containing Catalase and Superoxide Dismutase Activities Prepared by Polymerized Bovine Stroma-free Hemolysate
    Article Snippet: .. The following materials were purchased from different sources as shown: fresh bovine blood (McGill University, MacDonald campus), NaCl (Fisher), Potassium Phosphate (Sigma), Toluene (Sigma), Sodium Phosphate (Sigma), 99% Lysine Monohydrochloride (Sigma), 25% Glutaraldehyde (Sigma), 1.5cm*98cm Chromatography Column (Fisher), Sepacryl S-300HR (Sigma), Tris Base (Sigma), 37% Hydrochloric Acid (Sigma), Catalase from bovine liver (Sigma), Superoxide Dismuatase from bovine erythrocyte (Sigma), 30% Brij 35 solution (Sigma), Drabkin (Sigma), Molecular Weight Marker Kit (29,000–700,000) (Sigma), UltraPure Water (Cayman Chemical), 100kDa Centrifugal Filter (Millipore), 14.1 g/dl Haemiglobincyanide Standard (J.T. .. Baker), Catalase Assay Kit (Sigma), Superoxide Dismutase Kit (R & D Systems), 3%(w/w) Hydrogen Peroxide (Sigma), Sodium Perborate Tetrahydrate (Sigma), 95–98% Sulfuric Acid (Sigma), 99% Potassium Permanganate (Sigma), Hepalean 1000U.S.P. units/ml (Organon), Pentobarbital Sodique (CEVA Sante Animal), Complement C3a des Arg ELISA Kits (Assay Design), Sodium Hydroxide (Sigma), 99.9% Sodium Azide (Sigma), Agar (Sigma).

    Article Title: Preparation of SUMO proteases and kinetic analysis using endogenous substrates
    Article Snippet: .. To ensure reproducibility and to protect chromatography columns from undo wear and tear, all chromatographic steps were performed using filtered buffer solutions prepared from MilliQ (Millipore) water or the equivalent. ..

    Molecular Weight:

    Article Title: The Immunological Properties of Stroma-free Polyhemolysate Containing Catalase and Superoxide Dismutase Activities Prepared by Polymerized Bovine Stroma-free Hemolysate
    Article Snippet: .. The following materials were purchased from different sources as shown: fresh bovine blood (McGill University, MacDonald campus), NaCl (Fisher), Potassium Phosphate (Sigma), Toluene (Sigma), Sodium Phosphate (Sigma), 99% Lysine Monohydrochloride (Sigma), 25% Glutaraldehyde (Sigma), 1.5cm*98cm Chromatography Column (Fisher), Sepacryl S-300HR (Sigma), Tris Base (Sigma), 37% Hydrochloric Acid (Sigma), Catalase from bovine liver (Sigma), Superoxide Dismuatase from bovine erythrocyte (Sigma), 30% Brij 35 solution (Sigma), Drabkin (Sigma), Molecular Weight Marker Kit (29,000–700,000) (Sigma), UltraPure Water (Cayman Chemical), 100kDa Centrifugal Filter (Millipore), 14.1 g/dl Haemiglobincyanide Standard (J.T. .. Baker), Catalase Assay Kit (Sigma), Superoxide Dismutase Kit (R & D Systems), 3%(w/w) Hydrogen Peroxide (Sigma), Sodium Perborate Tetrahydrate (Sigma), 95–98% Sulfuric Acid (Sigma), 99% Potassium Permanganate (Sigma), Hepalean 1000U.S.P. units/ml (Organon), Pentobarbital Sodique (CEVA Sante Animal), Complement C3a des Arg ELISA Kits (Assay Design), Sodium Hydroxide (Sigma), 99.9% Sodium Azide (Sigma), Agar (Sigma).

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  • 88
    Millipore streptavidin sepharose affinity columns
    <t>Streptavidin-affinity-enriched</t> PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using <t>streptavidin—Sepharose</t> affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.
    Streptavidin Sepharose Affinity Columns, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore trna sepharose column
    In vitro production of Arabidopsis <t>tRNA</t> ligase and purification of the recombinant protein. Overexpression of the Arabidopsis tRNA ligase was accomplished in the RTS 100 wheat germ CECF system. Incubation of the vector DNA, carrying the tRNA ligase cDNA with a C-terminal histidine tag was performed in a 50 μl reaction mixture in the presence of [ 35 S]methionine for 24 h at 24°C. The overexpressed C-tagged tRNA ligase was subsequently purified by Ni-NTA chromatography. Aliquots of 2 μl of the total reaction mixture (lane a), of the flow-through (lane b) and wash fractions (lane c) after binding of the protein mixture to the Ni-NTA <t>agarose</t> as well as of the first fractions eluting with 500 mM imidazole (lanes d and e) were loaded onto a 7.5% polyacrylamide/0.1% SDS gel. After Coomassie brilliant blue staining (left panel), the gel was developed for fluorography (right panel) ( 61 ).
    Trna Sepharose Column, 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
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    94
    Millipore immunoblotting
    PP2A associates directly with Dvl2 and dephosphorylates Dvl2 . Panel A , probing interaction of Dvl2 and PP2A in vivo . Whole-cell extracts (2 mg) prepared from F9 cells expressing Rfz1 were incubated with immobilized GST gel alone or with GST-PP2A C-subunit-immobilized gel. Bound proteins were released and resolved by SDS-PAGE. The resolved proteins were subjected to blotting and stained with either with anti-PP2A C subunit antibodies ( top panel ) or anti-Dvl2 antibodies ( bottom panel ). Panel B , pull-downs of Dvl2 from F9 cells reveal associated PP2A. F9 cells lysates were subjected to analysis by pull-downs of either Dvl2 or of mouse IgG (as a control). The immune precipitates were subjected to SDS-PAGE, <t>immunoblotting,</t> and staining with either anti-Dvl2 antibodies (top panel) or anti-PP2A C-subunit antibodies (bottom panel). Panel C , Dvl2 domains structure. Panel D , direct association of Dvl2 and PP2A in vitro . Purified mouse PP2A enzyme (PP2A, AC subunit dimers) was incubated with one of four domains of Dvl2 engineered as a fusion protein with GST and then immobilized: immobilized GST-PDZ, GST-DIX, GST-DEP, GST-SH3 and GST itself (as a control) at 4°C for 1 hr. The bound proteins were separated by SDS-PAGE, blotted, and stained with either anti-PP2A C-subunit antibodies or anti-GST antibodies. Panel E , PP2A dephosphorylates phospho-rDvl2. 6-Histidinyl-tagged phosphorylated Dvl2 was expressed in Sf9 cell and purified by Ni-NTA column chromatography as described in Methods . Purified phospho-rDvl2 protein was incubated with either purified calf alkaline phosphatase or with purified PP2A for 1.5 hr. The incubation was terminated by addition of SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE, blotted, and stained with either anti-phospho-Ser antibodies ( top of panel ) or with anti-Dvl2 antibodies ( bottom of panel ). The immune complexes were made visible by use of an alkaline phosphatase-conjugated, second antibody and BCIP/NBT as substrates. The results shown are from a single experiment, duplicated with essentially similar results.
    Immunoblotting, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore anti bmp 7 pd
    Affinity purification of recombinant proteins used in this study. A , Coomassie Brilliant Blue-stained SDS-polyacrylamide quality control gels of recombinantly expressed and affinity-purified <t>BMP-7</t> PD variants and proteins representing the fibrillin-1 N terminus. B ( left ), size exclusion chromatogram of the BMP-7 PD-GF complex after chelating chromatography utilizing the His 6 tag placed at the N terminus of the PD. The chromatogram shows the BMP-7 PD-GF complex mainly eluting in one peak. Right , Coomassie Brilliant Blue-stained SDS-polyacrylamide gel showing the purity of the peak fraction. C , Coomassie Brilliant Blue-stained SDS-polyacrylamide gels showing successful separation of the GF from the PD. The separation was performed as described previously ( 20 ). BMP-7 complex was separated into BMP-7 PD (34 kDa) and GF dimer (31 kDa) after dialysis into 8 m urea followed by chelating chromatography, where the PD was bound to the affinity column, and the GF was obtained in the flow-through.
    Anti Bmp 7 Pd, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Streptavidin-affinity-enriched PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using streptavidin—Sepharose affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.

    Journal: PLoS ONE

    Article Title: Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

    doi: 10.1371/journal.pone.0133033

    Figure Lengend Snippet: Streptavidin-affinity-enriched PBI686-tagged protein extracts. Total protein extract, photo-cross-linked with PBI686 was enriched using streptavidin—Sepharose affinity chromatography. Eluted proteins were desalted, concentrated and analysed using far-Western blot analysis with a streptavidin—HRP conjugate (lane 1) and silver-staining (lane 2) techniques. Band regions that were excised are indicated with letters A-C. The molecular mass in kDa is indicated.

    Article Snippet: Protein fractions were eluted by streptavidin—Sepharose affinity columns, desalted, concentrated using AmiconTM Ultrafree centrifugal filters (Millipore), and visualized using a FOCUS-FAST silver-stain kit.

    Techniques: Affinity Chromatography, Far Western Blot, Silver Staining

    In vitro production of Arabidopsis tRNA ligase and purification of the recombinant protein. Overexpression of the Arabidopsis tRNA ligase was accomplished in the RTS 100 wheat germ CECF system. Incubation of the vector DNA, carrying the tRNA ligase cDNA with a C-terminal histidine tag was performed in a 50 μl reaction mixture in the presence of [ 35 S]methionine for 24 h at 24°C. The overexpressed C-tagged tRNA ligase was subsequently purified by Ni-NTA chromatography. Aliquots of 2 μl of the total reaction mixture (lane a), of the flow-through (lane b) and wash fractions (lane c) after binding of the protein mixture to the Ni-NTA agarose as well as of the first fractions eluting with 500 mM imidazole (lanes d and e) were loaded onto a 7.5% polyacrylamide/0.1% SDS gel. After Coomassie brilliant blue staining (left panel), the gel was developed for fluorography (right panel) ( 61 ).

    Journal: Nucleic Acids Research

    Article Title: Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

    doi: 10.1093/nar/gki174

    Figure Lengend Snippet: In vitro production of Arabidopsis tRNA ligase and purification of the recombinant protein. Overexpression of the Arabidopsis tRNA ligase was accomplished in the RTS 100 wheat germ CECF system. Incubation of the vector DNA, carrying the tRNA ligase cDNA with a C-terminal histidine tag was performed in a 50 μl reaction mixture in the presence of [ 35 S]methionine for 24 h at 24°C. The overexpressed C-tagged tRNA ligase was subsequently purified by Ni-NTA chromatography. Aliquots of 2 μl of the total reaction mixture (lane a), of the flow-through (lane b) and wash fractions (lane c) after binding of the protein mixture to the Ni-NTA agarose as well as of the first fractions eluting with 500 mM imidazole (lanes d and e) were loaded onto a 7.5% polyacrylamide/0.1% SDS gel. After Coomassie brilliant blue staining (left panel), the gel was developed for fluorography (right panel) ( 61 ).

    Article Snippet: The pooled fractions from the tRNA Sepharose column were concentrated by ultrafiltration with Microcon 100 (Millipore, Schwalbach, Germany) and applied to a 10% polyacrylamide/0.1% SDS gel ( ).

    Techniques: In Vitro, Purification, Recombinant, Over Expression, Incubation, Plasmid Preparation, Chromatography, Flow Cytometry, Binding Assay, SDS-Gel, Staining

    Isolation of wheat germ tRNA ligase. ( A ) Purification scheme. RNA ligase was purified from the soluble protein fraction of wheat embryos (S100 extract) by six consecutive steps. ( B ) As substrate for assaying tRNA ligase activity, we have used a natural modified Nicotiana pre-tRNA Tyr (NtY9-T7-M1). The arrows in the two 4 nt bulge loops indicate the 3′ and 5′ splice sites and dots identify the anticodon. ( C ) Fractionation of wheat germ tRNA ligase by Cibacron Blue Trisacryl M chromatography and ligation activity assay. Partially purified tRNA ligase from the Heparin Sepharose column was applied onto a Blue-Trisacryl M column. Elution of tRNA ligase was performed with a gradient of 150–800 mM KCl. Fractions of 5 ml were collected. Splicing endonuclease co-eluted with tRNA ligase at this purification step, generating 3′ and 5′ tRNA halves. Reaction mixtures (20 μl) contained 20 mM Tris–HCl, pH 7.5, 6 mM Mg(OAc) 2 , 80 μM spermine, 1 mM ATP, 0.5 mM GTP, 0.1 mM DTT, 0.5% Triton X-100, 40 fmol (4 × 10 4 c.p.m.) of T7-transcript (NtY9-T7-M1) and 2 μl from eluted fractions. Incubation was for 30 min at 37°C. Products were analysed on a 12.5% polyacrylamide/8 M urea gel. tRNA ligase activity elutes between 280 and 540 mM KCl as revealed by the detection of mature, spliced tRNA.

    Journal: Nucleic Acids Research

    Article Title: Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

    doi: 10.1093/nar/gki174

    Figure Lengend Snippet: Isolation of wheat germ tRNA ligase. ( A ) Purification scheme. RNA ligase was purified from the soluble protein fraction of wheat embryos (S100 extract) by six consecutive steps. ( B ) As substrate for assaying tRNA ligase activity, we have used a natural modified Nicotiana pre-tRNA Tyr (NtY9-T7-M1). The arrows in the two 4 nt bulge loops indicate the 3′ and 5′ splice sites and dots identify the anticodon. ( C ) Fractionation of wheat germ tRNA ligase by Cibacron Blue Trisacryl M chromatography and ligation activity assay. Partially purified tRNA ligase from the Heparin Sepharose column was applied onto a Blue-Trisacryl M column. Elution of tRNA ligase was performed with a gradient of 150–800 mM KCl. Fractions of 5 ml were collected. Splicing endonuclease co-eluted with tRNA ligase at this purification step, generating 3′ and 5′ tRNA halves. Reaction mixtures (20 μl) contained 20 mM Tris–HCl, pH 7.5, 6 mM Mg(OAc) 2 , 80 μM spermine, 1 mM ATP, 0.5 mM GTP, 0.1 mM DTT, 0.5% Triton X-100, 40 fmol (4 × 10 4 c.p.m.) of T7-transcript (NtY9-T7-M1) and 2 μl from eluted fractions. Incubation was for 30 min at 37°C. Products were analysed on a 12.5% polyacrylamide/8 M urea gel. tRNA ligase activity elutes between 280 and 540 mM KCl as revealed by the detection of mature, spliced tRNA.

    Article Snippet: The pooled fractions from the tRNA Sepharose column were concentrated by ultrafiltration with Microcon 100 (Millipore, Schwalbach, Germany) and applied to a 10% polyacrylamide/0.1% SDS gel ( ).

    Techniques: Isolation, Purification, Activity Assay, Modification, Fractionation, Chromatography, Ligation, Incubation

    Gel filtration on Superdex™ 200 and adenylyltransferase activity of wheat germ tRNA ligase. ( A ) Partially purified tRNA ligase from the Source S15 column was subjected to gel filtration on Superdex™ 200. The column (HiLoad™ 16/60) was run with a flow rate of 1 ml/min. Fractions of 2 ml were collected. Aliquots of the elution fraction were analysed on a 7.5% polyacrylamide/0.1% SDS gel. The proteins were visualized by silver staining. ( B ) Appropriate aliquots of the indicated fractions were incubated in the presence of [α- 32 P]ATP for 15 min at 37°C. The ligase–[ 32 P]AMP adduct was detected by autoradiography of the dried gel. ( C ) The peak fractions from the tRNA Sepharose column were concentrated by ultrafiltration and 1/20 of this material was applied onto a 10% polyacrylamide/0.1% SDS gel and stained with Coomassie blue for analytical valuation. The arrows point to the position of the RNA ligase protein with an approximate molecular weight of 125 kDa. Protein size standards in kDa are indicated on the right.

    Journal: Nucleic Acids Research

    Article Title: Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

    doi: 10.1093/nar/gki174

    Figure Lengend Snippet: Gel filtration on Superdex™ 200 and adenylyltransferase activity of wheat germ tRNA ligase. ( A ) Partially purified tRNA ligase from the Source S15 column was subjected to gel filtration on Superdex™ 200. The column (HiLoad™ 16/60) was run with a flow rate of 1 ml/min. Fractions of 2 ml were collected. Aliquots of the elution fraction were analysed on a 7.5% polyacrylamide/0.1% SDS gel. The proteins were visualized by silver staining. ( B ) Appropriate aliquots of the indicated fractions were incubated in the presence of [α- 32 P]ATP for 15 min at 37°C. The ligase–[ 32 P]AMP adduct was detected by autoradiography of the dried gel. ( C ) The peak fractions from the tRNA Sepharose column were concentrated by ultrafiltration and 1/20 of this material was applied onto a 10% polyacrylamide/0.1% SDS gel and stained with Coomassie blue for analytical valuation. The arrows point to the position of the RNA ligase protein with an approximate molecular weight of 125 kDa. Protein size standards in kDa are indicated on the right.

    Article Snippet: The pooled fractions from the tRNA Sepharose column were concentrated by ultrafiltration with Microcon 100 (Millipore, Schwalbach, Germany) and applied to a 10% polyacrylamide/0.1% SDS gel ( ).

    Techniques: Filtration, Activity Assay, Purification, Flow Cytometry, SDS-Gel, Silver Staining, Incubation, Autoradiography, Staining, Molecular Weight

    PP2A associates directly with Dvl2 and dephosphorylates Dvl2 . Panel A , probing interaction of Dvl2 and PP2A in vivo . Whole-cell extracts (2 mg) prepared from F9 cells expressing Rfz1 were incubated with immobilized GST gel alone or with GST-PP2A C-subunit-immobilized gel. Bound proteins were released and resolved by SDS-PAGE. The resolved proteins were subjected to blotting and stained with either with anti-PP2A C subunit antibodies ( top panel ) or anti-Dvl2 antibodies ( bottom panel ). Panel B , pull-downs of Dvl2 from F9 cells reveal associated PP2A. F9 cells lysates were subjected to analysis by pull-downs of either Dvl2 or of mouse IgG (as a control). The immune precipitates were subjected to SDS-PAGE, immunoblotting, and staining with either anti-Dvl2 antibodies (top panel) or anti-PP2A C-subunit antibodies (bottom panel). Panel C , Dvl2 domains structure. Panel D , direct association of Dvl2 and PP2A in vitro . Purified mouse PP2A enzyme (PP2A, AC subunit dimers) was incubated with one of four domains of Dvl2 engineered as a fusion protein with GST and then immobilized: immobilized GST-PDZ, GST-DIX, GST-DEP, GST-SH3 and GST itself (as a control) at 4°C for 1 hr. The bound proteins were separated by SDS-PAGE, blotted, and stained with either anti-PP2A C-subunit antibodies or anti-GST antibodies. Panel E , PP2A dephosphorylates phospho-rDvl2. 6-Histidinyl-tagged phosphorylated Dvl2 was expressed in Sf9 cell and purified by Ni-NTA column chromatography as described in Methods . Purified phospho-rDvl2 protein was incubated with either purified calf alkaline phosphatase or with purified PP2A for 1.5 hr. The incubation was terminated by addition of SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE, blotted, and stained with either anti-phospho-Ser antibodies ( top of panel ) or with anti-Dvl2 antibodies ( bottom of panel ). The immune complexes were made visible by use of an alkaline phosphatase-conjugated, second antibody and BCIP/NBT as substrates. The results shown are from a single experiment, duplicated with essentially similar results.

    Journal: Journal of Molecular Signaling

    Article Title: Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/?-catenin signaling

    doi: 10.1186/1750-2187-2-12

    Figure Lengend Snippet: PP2A associates directly with Dvl2 and dephosphorylates Dvl2 . Panel A , probing interaction of Dvl2 and PP2A in vivo . Whole-cell extracts (2 mg) prepared from F9 cells expressing Rfz1 were incubated with immobilized GST gel alone or with GST-PP2A C-subunit-immobilized gel. Bound proteins were released and resolved by SDS-PAGE. The resolved proteins were subjected to blotting and stained with either with anti-PP2A C subunit antibodies ( top panel ) or anti-Dvl2 antibodies ( bottom panel ). Panel B , pull-downs of Dvl2 from F9 cells reveal associated PP2A. F9 cells lysates were subjected to analysis by pull-downs of either Dvl2 or of mouse IgG (as a control). The immune precipitates were subjected to SDS-PAGE, immunoblotting, and staining with either anti-Dvl2 antibodies (top panel) or anti-PP2A C-subunit antibodies (bottom panel). Panel C , Dvl2 domains structure. Panel D , direct association of Dvl2 and PP2A in vitro . Purified mouse PP2A enzyme (PP2A, AC subunit dimers) was incubated with one of four domains of Dvl2 engineered as a fusion protein with GST and then immobilized: immobilized GST-PDZ, GST-DIX, GST-DEP, GST-SH3 and GST itself (as a control) at 4°C for 1 hr. The bound proteins were separated by SDS-PAGE, blotted, and stained with either anti-PP2A C-subunit antibodies or anti-GST antibodies. Panel E , PP2A dephosphorylates phospho-rDvl2. 6-Histidinyl-tagged phosphorylated Dvl2 was expressed in Sf9 cell and purified by Ni-NTA column chromatography as described in Methods . Purified phospho-rDvl2 protein was incubated with either purified calf alkaline phosphatase or with purified PP2A for 1.5 hr. The incubation was terminated by addition of SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE, blotted, and stained with either anti-phospho-Ser antibodies ( top of panel ) or with anti-Dvl2 antibodies ( bottom of panel ). The immune complexes were made visible by use of an alkaline phosphatase-conjugated, second antibody and BCIP/NBT as substrates. The results shown are from a single experiment, duplicated with essentially similar results.

    Article Snippet: Immunoblotting and quantification analysis of proteins Proteins (60–100 μg samples) were analyzed by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon membrane (Millipore, Bedford, MA).

    Techniques: In Vivo, Expressing, Incubation, SDS Page, Staining, In Vitro, Purification, Column Chromatography

    PP2A shuttles to the plasma membrane and nuclear subcellular fractions in response to Wnt3a . Cells expressing Rfz1 receptor were harvested at indicated time point after Wnt3a stimulation. Cells were fractionated to the plasma membrane (PM), cytoplasm (CY) and nuclei (NU), as described in the Methods . Subcelluar fractions obtained from control (time = 0) and Wnt3a-stimulated cells were analyzed by immunoblotting with anti-PP2A C-subunit antibody. PM (blue line), CY (pink line) and NU (green line) fractions are displayed. Stained protein bands were quantified and the values are presented as

    Journal: Journal of Molecular Signaling

    Article Title: Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/?-catenin signaling

    doi: 10.1186/1750-2187-2-12

    Figure Lengend Snippet: PP2A shuttles to the plasma membrane and nuclear subcellular fractions in response to Wnt3a . Cells expressing Rfz1 receptor were harvested at indicated time point after Wnt3a stimulation. Cells were fractionated to the plasma membrane (PM), cytoplasm (CY) and nuclei (NU), as described in the Methods . Subcelluar fractions obtained from control (time = 0) and Wnt3a-stimulated cells were analyzed by immunoblotting with anti-PP2A C-subunit antibody. PM (blue line), CY (pink line) and NU (green line) fractions are displayed. Stained protein bands were quantified and the values are presented as "fold of zero time point". Bottom three panels of immunoblots are stained with antibodies against the following subcellular fraction marker proteins: Na + -K + -ATPase (plasma membrane), GAPDH (cytoplasm) and fibrillarin (nuclei), respectively. The results are shown as mean values ± S.E. from 5 or more independent experiments. Representative blots are displayed, as is the quantitative analysis of cellular content extracted from a compilation of data from the separate experiments (graphs).

    Article Snippet: Immunoblotting and quantification analysis of proteins Proteins (60–100 μg samples) were analyzed by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon membrane (Millipore, Bedford, MA).

    Techniques: Expressing, Staining, Western Blot, Marker

    Dvl2 interacts with PP2A and PP2A activity is attenuated in response to Wnt3a . Panel A , Association of Dvl2 and PP2A was investigated in the cytosolic fraction. Cell lysates (1 mg) were immunoprecipitated with Anti-Dvl2 antibody. Bound proteins were resolved by SDS-PAGE, immunoblotting, and made visible by staining with either anti-Dvl2 or anti-PP2A C-subunit antibodies. Dvl2 and PP2AC association is displayed as

    Journal: Journal of Molecular Signaling

    Article Title: Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/?-catenin signaling

    doi: 10.1186/1750-2187-2-12

    Figure Lengend Snippet: Dvl2 interacts with PP2A and PP2A activity is attenuated in response to Wnt3a . Panel A , Association of Dvl2 and PP2A was investigated in the cytosolic fraction. Cell lysates (1 mg) were immunoprecipitated with Anti-Dvl2 antibody. Bound proteins were resolved by SDS-PAGE, immunoblotting, and made visible by staining with either anti-Dvl2 or anti-PP2A C-subunit antibodies. Dvl2 and PP2AC association is displayed as "fold" (time = 0, set to "1"). Representative blots are displayed. The results are shown as mean values ± S.E. from 5 independent experiments. Panel B , F9 cells expressing Rfz1 were stimulated with purified Wnt3a for the indicated time. Cells were washed with PBS and lysed. Cell lysates were applied to a small Sephadex G-50 column to remove small molecular substances that interfere with the assay, and PP2A activity determined. Results were shown as the mean values ± S.E. from 8 independent experiments.

    Article Snippet: Immunoblotting and quantification analysis of proteins Proteins (60–100 μg samples) were analyzed by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon membrane (Millipore, Bedford, MA).

    Techniques: Activity Assay, Immunoprecipitation, SDS Page, Staining, Expressing, Purification

    Suppression of PP2A activity by siRNA or small t antigen enhances Lef/Tcf-sensitive transcription . Panel A , Rfz1-expressing F9 cells were treated with OA for 1 hr or siRNA targeting PP2A C subunit for 48 hr, or co-expression of small t antigen for 48 hr. Cell lysates were applied to a small Sephadex-G50 column and PP2A activity assay was carried out using pNPP as a substrate. The results are shown as mean values ± S.E. from 8–10 independent experiments. Abundance of PP2A C subunit was determined by Western immunoblotting with anti-PP2A C subunit antibody. Panel B , cells were treated with siRNAs targeting the PP2A C-subunit for one day before co-transfection of the cells with Rfz1 and Super8xpTOPFlash plasmids. Cells were stimulated with or without Wnt 3a for 8 hr. The luciferase gene reporter was assayed and is displayed relative to the unstimulated cells (set to

    Journal: Journal of Molecular Signaling

    Article Title: Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/?-catenin signaling

    doi: 10.1186/1750-2187-2-12

    Figure Lengend Snippet: Suppression of PP2A activity by siRNA or small t antigen enhances Lef/Tcf-sensitive transcription . Panel A , Rfz1-expressing F9 cells were treated with OA for 1 hr or siRNA targeting PP2A C subunit for 48 hr, or co-expression of small t antigen for 48 hr. Cell lysates were applied to a small Sephadex-G50 column and PP2A activity assay was carried out using pNPP as a substrate. The results are shown as mean values ± S.E. from 8–10 independent experiments. Abundance of PP2A C subunit was determined by Western immunoblotting with anti-PP2A C subunit antibody. Panel B , cells were treated with siRNAs targeting the PP2A C-subunit for one day before co-transfection of the cells with Rfz1 and Super8xpTOPFlash plasmids. Cells were stimulated with or without Wnt 3a for 8 hr. The luciferase gene reporter was assayed and is displayed relative to the unstimulated cells (set to "1"). The results showed mean values ± S.E., obtained from five separate experiments. Statistical significance is indicated (*, p

    Article Snippet: Immunoblotting and quantification analysis of proteins Proteins (60–100 μg samples) were analyzed by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon membrane (Millipore, Bedford, MA).

    Techniques: Activity Assay, Expressing, Western Blot, Cotransfection, Luciferase

    Suppression of PP2A activity alters cellular abundant of Wnt/β-catenin signaling elements . F9 cells expressing Rfz1 were either pretreated with OA for 1 hr or treated with siRNA targeting PP2A C- subunit for 48 hr, or transfected to express SV40 small t antigen for 48 hr. Cells were washed with PBS twice and lysed. Cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting, blots stained with anti-Axin, anti-β-catenin, anti-Dvl2, anti-GSK3β, anti-p-Ser (9)-GSK3β, anti-PP2A C or anti-GAPDH antibody. The relative amounts of the proteins in each fraction were established by densitometry, as described in Methods . The relative abundance of each signaling molecule in the untreated cell, whole-cell extract was set to

    Journal: Journal of Molecular Signaling

    Article Title: Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/?-catenin signaling

    doi: 10.1186/1750-2187-2-12

    Figure Lengend Snippet: Suppression of PP2A activity alters cellular abundant of Wnt/β-catenin signaling elements . F9 cells expressing Rfz1 were either pretreated with OA for 1 hr or treated with siRNA targeting PP2A C- subunit for 48 hr, or transfected to express SV40 small t antigen for 48 hr. Cells were washed with PBS twice and lysed. Cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting, blots stained with anti-Axin, anti-β-catenin, anti-Dvl2, anti-GSK3β, anti-p-Ser (9)-GSK3β, anti-PP2A C or anti-GAPDH antibody. The relative amounts of the proteins in each fraction were established by densitometry, as described in Methods . The relative abundance of each signaling molecule in the untreated cell, whole-cell extract was set to "1". The results are shown as mean values ± S.E. from 8–10 independent experiments. Right-handed panel displays the representative immunoblots, stained for Axin, β-catenin, GSK3β and p-Ser (9)-GSK3β.

    Article Snippet: Immunoblotting and quantification analysis of proteins Proteins (60–100 μg samples) were analyzed by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon membrane (Millipore, Bedford, MA).

    Techniques: Activity Assay, Expressing, Transfection, SDS Page, Staining, Western Blot

    Effects of inhibition of PP2A on cellular abundance of Wnt/β-catenin signaling elements . Graphs display the relative cellular abundance of Wnt/β-catenin signaling elements in cells following stimulation with Wnt3a. F9 cells expressing Rfz1 were untreated or treated with Wnt3a in the presence or absence of OA for 0 to 90 min. For OA treatment, F9 cells expressing Rfz1 were treated with OA for 1 hr prior to Wnt3a stimulation. For small t antigen experiments, F9 cells were transiently co-transfected with Rfz1 and small t antigen, and then stimulated with Wnt3a for the indicated times. Cells were collected and lysed. Lysates (60–100 μg protein) were subjected to SDS-PAGE and analyzed by immunoblotting, blots stained with antibodies targeting the signaling molecules indicated. Bands were quantified by densitometry, as described in Experimental Procedure and values are displayed as

    Journal: Journal of Molecular Signaling

    Article Title: Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/?-catenin signaling

    doi: 10.1186/1750-2187-2-12

    Figure Lengend Snippet: Effects of inhibition of PP2A on cellular abundance of Wnt/β-catenin signaling elements . Graphs display the relative cellular abundance of Wnt/β-catenin signaling elements in cells following stimulation with Wnt3a. F9 cells expressing Rfz1 were untreated or treated with Wnt3a in the presence or absence of OA for 0 to 90 min. For OA treatment, F9 cells expressing Rfz1 were treated with OA for 1 hr prior to Wnt3a stimulation. For small t antigen experiments, F9 cells were transiently co-transfected with Rfz1 and small t antigen, and then stimulated with Wnt3a for the indicated times. Cells were collected and lysed. Lysates (60–100 μg protein) were subjected to SDS-PAGE and analyzed by immunoblotting, blots stained with antibodies targeting the signaling molecules indicated. Bands were quantified by densitometry, as described in Experimental Procedure and values are displayed as "fold", with time = 0 set as "1". The results are shown as mean values ± S.E. from 6–8 independent experiments. Representative blots are displayed, as is the quantitative analysis of cellular content extracted from 6–8 separate experiments (graphs).

    Article Snippet: Immunoblotting and quantification analysis of proteins Proteins (60–100 μg samples) were analyzed by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon membrane (Millipore, Bedford, MA).

    Techniques: Inhibition, Expressing, Transfection, SDS Page, Staining

    Affinity purification of recombinant proteins used in this study. A , Coomassie Brilliant Blue-stained SDS-polyacrylamide quality control gels of recombinantly expressed and affinity-purified BMP-7 PD variants and proteins representing the fibrillin-1 N terminus. B ( left ), size exclusion chromatogram of the BMP-7 PD-GF complex after chelating chromatography utilizing the His 6 tag placed at the N terminus of the PD. The chromatogram shows the BMP-7 PD-GF complex mainly eluting in one peak. Right , Coomassie Brilliant Blue-stained SDS-polyacrylamide gel showing the purity of the peak fraction. C , Coomassie Brilliant Blue-stained SDS-polyacrylamide gels showing successful separation of the GF from the PD. The separation was performed as described previously ( 20 ). BMP-7 complex was separated into BMP-7 PD (34 kDa) and GF dimer (31 kDa) after dialysis into 8 m urea followed by chelating chromatography, where the PD was bound to the affinity column, and the GF was obtained in the flow-through.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1 *

    doi: 10.1074/jbc.M115.704734

    Figure Lengend Snippet: Affinity purification of recombinant proteins used in this study. A , Coomassie Brilliant Blue-stained SDS-polyacrylamide quality control gels of recombinantly expressed and affinity-purified BMP-7 PD variants and proteins representing the fibrillin-1 N terminus. B ( left ), size exclusion chromatogram of the BMP-7 PD-GF complex after chelating chromatography utilizing the His 6 tag placed at the N terminus of the PD. The chromatogram shows the BMP-7 PD-GF complex mainly eluting in one peak. Right , Coomassie Brilliant Blue-stained SDS-polyacrylamide gel showing the purity of the peak fraction. C , Coomassie Brilliant Blue-stained SDS-polyacrylamide gels showing successful separation of the GF from the PD. The separation was performed as described previously ( 20 ). BMP-7 complex was separated into BMP-7 PD (34 kDa) and GF dimer (31 kDa) after dialysis into 8 m urea followed by chelating chromatography, where the PD was bound to the affinity column, and the GF was obtained in the flow-through.

    Article Snippet: Each gradient was collected in 28 fractions and analyzed by Western blotting using specific anti-BMP-7 PD (mAb2 from Millipore, Billerica, MA) and GF (500-P198, Peprotech) antibodies ( ).

    Techniques: Affinity Purification, Recombinant, Staining, Chromatography, Affinity Column, Flow Cytometry

    Interaction studies to identify the fibrillin-1 binding motif within the BMP-7 PD. A , domain structure of the N-terminal fibrillin-1 fragment used in the interaction study. B , sensorgrams of SPR binding studies suggesting that the fibrillin-1 binding domain within the BMP-7 PD resides within residues Gly 74 –Phe 185 . 0–80 n m rF87, containing the N-terminal unique domain, the first three EGF-like domains, the first hybrid motif, and the first two calcium-binding EGF- like domains of fibrillin-1, was injected onto immobilized BMP-7 PD variants. All injections were performed in HBS-EP buffer.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1 *

    doi: 10.1074/jbc.M115.704734

    Figure Lengend Snippet: Interaction studies to identify the fibrillin-1 binding motif within the BMP-7 PD. A , domain structure of the N-terminal fibrillin-1 fragment used in the interaction study. B , sensorgrams of SPR binding studies suggesting that the fibrillin-1 binding domain within the BMP-7 PD resides within residues Gly 74 –Phe 185 . 0–80 n m rF87, containing the N-terminal unique domain, the first three EGF-like domains, the first hybrid motif, and the first two calcium-binding EGF- like domains of fibrillin-1, was injected onto immobilized BMP-7 PD variants. All injections were performed in HBS-EP buffer.

    Article Snippet: Each gradient was collected in 28 fractions and analyzed by Western blotting using specific anti-BMP-7 PD (mAb2 from Millipore, Billerica, MA) and GF (500-P198, Peprotech) antibodies ( ).

    Techniques: Binding Assay, SPR Assay, Injection

    Identification of the GF binding motif within the BMP-7 PD. A , solid phase binding ELISA-style assays with immobilized BMP-7 PD truncation variants and GF in solution. B , SPR binding studies of BMP-7 N-terminal PD truncation variants and BMP-7 GF. Top panel , GF binding to the PD was robust to the presence of 1 m urea and pH reduction to 4.5 (full-length PD(30–292) immobilized, GF in solution). Bottom panels , GF was immobilized, and PD variants were injected in solution. C , BMP-7 reconstitution after dialysis of PD variants and GF. Successful reconstitution was monitored in a sandwich ELISA using an anti-His 6 antibody against the C-terminal His 6 tag on the PD as capture and a polyclonal anti-BMP-7 GF antibody as detector. Error bars , S.D. from three independent experiments. D , sequence alignment using ClustalW identifies the 65 PHRP 68 motif ( red ) as conserved within the BMP-5, -6, -7 subgroup of the TGF-β superfamily. Blue , predicted Ile 58 -Leu 59 -Leu 62 -Leu 64 GF binding motif ( 35 ). E , CD spectra of systematic truncation variants between Arg 67 and Pro 69 . Deletion of Pro 65 -His 66 results in a significant increase of α-helical content of 8% when compared with the 65–292 PD variant ( Table 1 ). This increase returned to normal levels upon stepwise N-terminal truncation of the subsequent two residues. The point mutation P65A resulted in a 4% increase of α-helical content ( Table 1 ), whereas H66A or P68A resulted in no or little change. Additional mutation of the subsequent three residues resulted in no additional change in α-helical content in the quadruple mutant variant P65A/H66A/R67A/P68A ( Table 1 ).

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1 *

    doi: 10.1074/jbc.M115.704734

    Figure Lengend Snippet: Identification of the GF binding motif within the BMP-7 PD. A , solid phase binding ELISA-style assays with immobilized BMP-7 PD truncation variants and GF in solution. B , SPR binding studies of BMP-7 N-terminal PD truncation variants and BMP-7 GF. Top panel , GF binding to the PD was robust to the presence of 1 m urea and pH reduction to 4.5 (full-length PD(30–292) immobilized, GF in solution). Bottom panels , GF was immobilized, and PD variants were injected in solution. C , BMP-7 reconstitution after dialysis of PD variants and GF. Successful reconstitution was monitored in a sandwich ELISA using an anti-His 6 antibody against the C-terminal His 6 tag on the PD as capture and a polyclonal anti-BMP-7 GF antibody as detector. Error bars , S.D. from three independent experiments. D , sequence alignment using ClustalW identifies the 65 PHRP 68 motif ( red ) as conserved within the BMP-5, -6, -7 subgroup of the TGF-β superfamily. Blue , predicted Ile 58 -Leu 59 -Leu 62 -Leu 64 GF binding motif ( 35 ). E , CD spectra of systematic truncation variants between Arg 67 and Pro 69 . Deletion of Pro 65 -His 66 results in a significant increase of α-helical content of 8% when compared with the 65–292 PD variant ( Table 1 ). This increase returned to normal levels upon stepwise N-terminal truncation of the subsequent two residues. The point mutation P65A resulted in a 4% increase of α-helical content ( Table 1 ), whereas H66A or P68A resulted in no or little change. Additional mutation of the subsequent three residues resulted in no additional change in α-helical content in the quadruple mutant variant P65A/H66A/R67A/P68A ( Table 1 ).

    Article Snippet: Each gradient was collected in 28 fractions and analyzed by Western blotting using specific anti-BMP-7 PD (mAb2 from Millipore, Billerica, MA) and GF (500-P198, Peprotech) antibodies ( ).

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Injection, Sandwich ELISA, Sequencing, Variant Assay, Mutagenesis

    SAXS data collected for BMP-7 complex. A , x-ray scattering profile of BMP-7 complex showing intensity as a function of q ( gray triangles ) and Gnom fit to the data ( black line ). Inset , Guinier plot showing R g of 4.8 nm. B , P ( r ) plot showing D max of 16 nm. C , Kratky plot showing profile typical of a folded protein. D , plateau in the Porod-Debye plot indicative of a non-flexible protein. E , ab initio models generated from SAXS data using GASBOR with 2-fold symmetry ( blue ); the averaged model is shown along with three representative models.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1 *

    doi: 10.1074/jbc.M115.704734

    Figure Lengend Snippet: SAXS data collected for BMP-7 complex. A , x-ray scattering profile of BMP-7 complex showing intensity as a function of q ( gray triangles ) and Gnom fit to the data ( black line ). Inset , Guinier plot showing R g of 4.8 nm. B , P ( r ) plot showing D max of 16 nm. C , Kratky plot showing profile typical of a folded protein. D , plateau in the Porod-Debye plot indicative of a non-flexible protein. E , ab initio models generated from SAXS data using GASBOR with 2-fold symmetry ( blue ); the averaged model is shown along with three representative models.

    Article Snippet: Each gradient was collected in 28 fractions and analyzed by Western blotting using specific anti-BMP-7 PD (mAb2 from Millipore, Billerica, MA) and GF (500-P198, Peprotech) antibodies ( ).

    Techniques: Generated

    Three-dimensional EM and solution SAXS models of BMP-7 PD-GF complex. Three-dimensional structure of BMP-7 complex was generated using TEM. A ( top ), representative electron micrograph of BMP-7 complex molecules ( scale bar , 100 nm); bottom , 12 images selected from 140 class sum images of 9,000 particles that represent different views of BMP-7 complex ( box size = 29.4 × 29.4 nm). B , class sum images were used to generate a three-dimensional TEM model of BMP-7 complex with 2-fold symmetry using angular reconstitution. C , superimposition of the BMP-9 complex structure ( 33 ) at 20 Å with the determined BMP-7 EM envelope suggests that the angle between the boomerang arms may be wider in BMP-9.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1 *

    doi: 10.1074/jbc.M115.704734

    Figure Lengend Snippet: Three-dimensional EM and solution SAXS models of BMP-7 PD-GF complex. Three-dimensional structure of BMP-7 complex was generated using TEM. A ( top ), representative electron micrograph of BMP-7 complex molecules ( scale bar , 100 nm); bottom , 12 images selected from 140 class sum images of 9,000 particles that represent different views of BMP-7 complex ( box size = 29.4 × 29.4 nm). B , class sum images were used to generate a three-dimensional TEM model of BMP-7 complex with 2-fold symmetry using angular reconstitution. C , superimposition of the BMP-9 complex structure ( 33 ) at 20 Å with the determined BMP-7 EM envelope suggests that the angle between the boomerang arms may be wider in BMP-9.

    Article Snippet: Each gradient was collected in 28 fractions and analyzed by Western blotting using specific anti-BMP-7 PD (mAb2 from Millipore, Billerica, MA) and GF (500-P198, Peprotech) antibodies ( ).

    Techniques: Generated, Transmission Electron Microscopy

    The 65 PHRP 68 motif within the N-terminal region of the BMP-7 PD is required for competition with the BMP type II receptor for GF binding. A , scheme of the experimental set-up. Full-length BMP-7 PD (residues 30–292) and the N-terminal truncation variant 65–292 were immobilized, and 100 n m BMP-7 GF was injected in the presence of 0–500 n m BMPRII receptor extracellular domain onto the chip first, followed by a second injection of 100 n m mAb anti-BMP-7 GF antibody to detect bound GF (all injections were in HBS-EP buffer). B , sensorgrams of 100 n m injected mAb anti-BMP-7 GF antibody to detect bound GF. C , increasing amounts of receptor resulted in comparable inhibition of GF binding to both immobilized PD variants, suggesting that the presence of the 65 PHRP 68 motif in 65–292 is responsible for PD competition with the type II receptor for the same binding site on the GF. Error bars , S.D. from three independent experiments. The schematic shows GF ( orange ) and type II receptor ( blue ) binding sites.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1 *

    doi: 10.1074/jbc.M115.704734

    Figure Lengend Snippet: The 65 PHRP 68 motif within the N-terminal region of the BMP-7 PD is required for competition with the BMP type II receptor for GF binding. A , scheme of the experimental set-up. Full-length BMP-7 PD (residues 30–292) and the N-terminal truncation variant 65–292 were immobilized, and 100 n m BMP-7 GF was injected in the presence of 0–500 n m BMPRII receptor extracellular domain onto the chip first, followed by a second injection of 100 n m mAb anti-BMP-7 GF antibody to detect bound GF (all injections were in HBS-EP buffer). B , sensorgrams of 100 n m injected mAb anti-BMP-7 GF antibody to detect bound GF. C , increasing amounts of receptor resulted in comparable inhibition of GF binding to both immobilized PD variants, suggesting that the presence of the 65 PHRP 68 motif in 65–292 is responsible for PD competition with the type II receptor for the same binding site on the GF. Error bars , S.D. from three independent experiments. The schematic shows GF ( orange ) and type II receptor ( blue ) binding sites.

    Article Snippet: Each gradient was collected in 28 fractions and analyzed by Western blotting using specific anti-BMP-7 PD (mAb2 from Millipore, Billerica, MA) and GF (500-P198, Peprotech) antibodies ( ).

    Techniques: Binding Assay, Variant Assay, Injection, Chromatin Immunoprecipitation, Inhibition

    Homology models of the BMP-7 complex and model of extracellular control of BMP GF activity via PD interactions with fibrillin-1 microfibrils. A ( top ), in the unbound, bioactive state, the BMP-7 complex adopts an open V-like shape. In this conformation, the PDs are in contact with each other via the first 18 N-terminal residues ( green ). The GF shows an extended, open conformation similar to the TGF-β-1 GF in the SLC ( 34 ), which enables positioning of the α1-helix ( purple rod ) of the PD within a pocket of the GF. The PD contains a 65 PHRP 68 motif ( red hinge ) located between the α1- and α2-helix ( red rod ), which serves as an important “molecular clamp” for maintaining interaction with the GF and is therefore required for proper PD competition with type II receptor binding. In this conformation, the α2-helix is not occupying the type II receptor binding site on the GF. Bottom , upon binding to fibrillin-1, the BMP-7 complex undergoes a conformational change. In this latent, closed conformation, the two PD arms may interact with each other via unmasked C-terminal self-interaction epitopes, which in turn facilitate the ring closure. In the closed ring shape conformation, the α2 occupies the type II receptor binding site, which confers latency to the GF. B , in solution, binding of type II receptors to the GF moiety of the BMP-7 complex results in displacement of the PDs as a dimer. The PDs remain tethered to each other via their N-terminal self-interaction epitopes ( green ). Binding to fibrillin-1 microfibrils ( green ) induces a conformational change within the PD that enables a closed ring-shaped conformation of the BMP-7 complex, rendering the GF latent. Homology models of the BMP-7 complex in its open and closed forms were generated using the structure of the TGF-β-1 SLC ( 34 ) and fitted into the shapes determined by TEM. For the open BMP-7 form, the model is fitted in the electron density map from EM, and for the closed form the model is shown as electron density rendered at 20 Å resolution. Orange , GF dimer; yellow circle , type II receptor binding site; green , N-terminal self-interaction epitope; magenta , α1-helix; red , α2-helix; red , stretch connecting α1- and α2-helix containing the 65 PHRP 68 motif; light blue , C-terminal portion of BMP-7 PD.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1 *

    doi: 10.1074/jbc.M115.704734

    Figure Lengend Snippet: Homology models of the BMP-7 complex and model of extracellular control of BMP GF activity via PD interactions with fibrillin-1 microfibrils. A ( top ), in the unbound, bioactive state, the BMP-7 complex adopts an open V-like shape. In this conformation, the PDs are in contact with each other via the first 18 N-terminal residues ( green ). The GF shows an extended, open conformation similar to the TGF-β-1 GF in the SLC ( 34 ), which enables positioning of the α1-helix ( purple rod ) of the PD within a pocket of the GF. The PD contains a 65 PHRP 68 motif ( red hinge ) located between the α1- and α2-helix ( red rod ), which serves as an important “molecular clamp” for maintaining interaction with the GF and is therefore required for proper PD competition with type II receptor binding. In this conformation, the α2-helix is not occupying the type II receptor binding site on the GF. Bottom , upon binding to fibrillin-1, the BMP-7 complex undergoes a conformational change. In this latent, closed conformation, the two PD arms may interact with each other via unmasked C-terminal self-interaction epitopes, which in turn facilitate the ring closure. In the closed ring shape conformation, the α2 occupies the type II receptor binding site, which confers latency to the GF. B , in solution, binding of type II receptors to the GF moiety of the BMP-7 complex results in displacement of the PDs as a dimer. The PDs remain tethered to each other via their N-terminal self-interaction epitopes ( green ). Binding to fibrillin-1 microfibrils ( green ) induces a conformational change within the PD that enables a closed ring-shaped conformation of the BMP-7 complex, rendering the GF latent. Homology models of the BMP-7 complex in its open and closed forms were generated using the structure of the TGF-β-1 SLC ( 34 ) and fitted into the shapes determined by TEM. For the open BMP-7 form, the model is fitted in the electron density map from EM, and for the closed form the model is shown as electron density rendered at 20 Å resolution. Orange , GF dimer; yellow circle , type II receptor binding site; green , N-terminal self-interaction epitope; magenta , α1-helix; red , α2-helix; red , stretch connecting α1- and α2-helix containing the 65 PHRP 68 motif; light blue , C-terminal portion of BMP-7 PD.

    Article Snippet: Each gradient was collected in 28 fractions and analyzed by Western blotting using specific anti-BMP-7 PD (mAb2 from Millipore, Billerica, MA) and GF (500-P198, Peprotech) antibodies ( ).

    Techniques: Activity Assay, Binding Assay, Generated, Transmission Electron Microscopy

    Binding to fibrillin-1 induces a conformational change of the BMP-7 complex, resulting in GF inhibition. A , domain structure of fibrillin-1 and used variants. B , BMP activity assay with BMP-7 complex captured via PD interactions, mAb against the N-terminal His 6 tag, or the N-terminal half of fibrillin-1 (rF11). C2C12 cells were seeded onto immobilized BMP-7 complex, and Id3 expression was measured as a read-out for BMP activity. Shown is dot blotting analysis of stripped BMP-7 complex by comparison with a diluted series of dots containing BMP-7 complex at known concentrations. The schematic depicts the different ways BMP-7 PD-GF complex is presented to the reporter cells. Antibody capture of the N-terminally placed His 6 tag on the PD ( green ) does not affect bioactivity; however, binding of fibrillin-1 within the PD ( blue ) induces a conformational change into a ring shape that confers latency. Orange , GF dimer; yellow circle , type II receptor binding site; green , N-terminal His 6 tag; magenta , α1-helix; red , α2-helix; red , stretch connecting α1- and α2-helix containing the 65 PHRP 68 motif; light blue , C-terminal portion of BMP-7 PD. C , dialyzing the small fibrillin-1 N-terminal fragment FUN to BMP-7 complex resulted in the formation of ring shapes and open intermediates that were absent in the BMP-7 complex-only sample ( Fig. 2 A ). Shown are a representative TEM electron micrograph ( scale bar , 100 nm) and 12 from 100 class averages of 11,000 particles ( box size , 28 × 28 nm). The small fibrillin-1 fragment FUN was not distinguishable from the background.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1 *

    doi: 10.1074/jbc.M115.704734

    Figure Lengend Snippet: Binding to fibrillin-1 induces a conformational change of the BMP-7 complex, resulting in GF inhibition. A , domain structure of fibrillin-1 and used variants. B , BMP activity assay with BMP-7 complex captured via PD interactions, mAb against the N-terminal His 6 tag, or the N-terminal half of fibrillin-1 (rF11). C2C12 cells were seeded onto immobilized BMP-7 complex, and Id3 expression was measured as a read-out for BMP activity. Shown is dot blotting analysis of stripped BMP-7 complex by comparison with a diluted series of dots containing BMP-7 complex at known concentrations. The schematic depicts the different ways BMP-7 PD-GF complex is presented to the reporter cells. Antibody capture of the N-terminally placed His 6 tag on the PD ( green ) does not affect bioactivity; however, binding of fibrillin-1 within the PD ( blue ) induces a conformational change into a ring shape that confers latency. Orange , GF dimer; yellow circle , type II receptor binding site; green , N-terminal His 6 tag; magenta , α1-helix; red , α2-helix; red , stretch connecting α1- and α2-helix containing the 65 PHRP 68 motif; light blue , C-terminal portion of BMP-7 PD. C , dialyzing the small fibrillin-1 N-terminal fragment FUN to BMP-7 complex resulted in the formation of ring shapes and open intermediates that were absent in the BMP-7 complex-only sample ( Fig. 2 A ). Shown are a representative TEM electron micrograph ( scale bar , 100 nm) and 12 from 100 class averages of 11,000 particles ( box size , 28 × 28 nm). The small fibrillin-1 fragment FUN was not distinguishable from the background.

    Article Snippet: Each gradient was collected in 28 fractions and analyzed by Western blotting using specific anti-BMP-7 PD (mAb2 from Millipore, Billerica, MA) and GF (500-P198, Peprotech) antibodies ( ).

    Techniques: Binding Assay, Inhibition, Activity Assay, Expressing, Transmission Electron Microscopy

    BMP-7 PD interacts with itself. A , dialysis of BMP-7 complex into 0.25–4 m urea reveals the presence of PD dimers monitored by velocity sedimentation experiments using 5–20% sucrose gradients. Each gradient was collected in 28 fractions (fraction 1 at 5% and fraction 28 at 20% sucrose) and subjected to Western blotting analysis for BMP-7 complex components. Western blots were incubated with anti-BMP-7 PD antibody first, stripped, and subsequently re-incubated with anti-BMP-7 GF antibody. B , SPR sensorgrams of self-interaction studies with BMP-7 full-length PD, 48–292, and 55–292, respectively, of immobilized full-length PD and PD variants representing the C-terminal end. C , BMP-7 complex reconstitution is affected by 30% upon deletion of the N-terminal PD self-interaction site. Error bars , mean ± S.D. from three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1 *

    doi: 10.1074/jbc.M115.704734

    Figure Lengend Snippet: BMP-7 PD interacts with itself. A , dialysis of BMP-7 complex into 0.25–4 m urea reveals the presence of PD dimers monitored by velocity sedimentation experiments using 5–20% sucrose gradients. Each gradient was collected in 28 fractions (fraction 1 at 5% and fraction 28 at 20% sucrose) and subjected to Western blotting analysis for BMP-7 complex components. Western blots were incubated with anti-BMP-7 PD antibody first, stripped, and subsequently re-incubated with anti-BMP-7 GF antibody. B , SPR sensorgrams of self-interaction studies with BMP-7 full-length PD, 48–292, and 55–292, respectively, of immobilized full-length PD and PD variants representing the C-terminal end. C , BMP-7 complex reconstitution is affected by 30% upon deletion of the N-terminal PD self-interaction site. Error bars , mean ± S.D. from three independent experiments.

    Article Snippet: Each gradient was collected in 28 fractions and analyzed by Western blotting using specific anti-BMP-7 PD (mAb2 from Millipore, Billerica, MA) and GF (500-P198, Peprotech) antibodies ( ).

    Techniques: Sedimentation, Western Blot, Incubation, SPR Assay

    Secondary structure analysis of BMP-7 PD. A , CD measurements of N-terminal BMP-7 PD truncation variants ( red ) in comparison with full-length BMP-7 PD (30–292, blue ). Bottom panel , middle , truncation of the first 43 residues in a shorter variant covering the first 184 N-terminal residues ( red ) results in significant reduction of the α-helical peak at 209 nm compared with the control ( blue ). Construct 166–217 suggests the existence of a third α-helix within this region. Secondary structure percentage calculated from these CD curves is listed in Tables 1 and 2 . B , secondary structure prediction based on CD measurements shows the position of α-helices ( red ). The position of β-sheets ( blue ) was guided by predicted secondary structure elements (PSIPRED) marked below the sequence ( yellow , α-helical regions; light blue , β-sheets).

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1 *

    doi: 10.1074/jbc.M115.704734

    Figure Lengend Snippet: Secondary structure analysis of BMP-7 PD. A , CD measurements of N-terminal BMP-7 PD truncation variants ( red ) in comparison with full-length BMP-7 PD (30–292, blue ). Bottom panel , middle , truncation of the first 43 residues in a shorter variant covering the first 184 N-terminal residues ( red ) results in significant reduction of the α-helical peak at 209 nm compared with the control ( blue ). Construct 166–217 suggests the existence of a third α-helix within this region. Secondary structure percentage calculated from these CD curves is listed in Tables 1 and 2 . B , secondary structure prediction based on CD measurements shows the position of α-helices ( red ). The position of β-sheets ( blue ) was guided by predicted secondary structure elements (PSIPRED) marked below the sequence ( yellow , α-helical regions; light blue , β-sheets).

    Article Snippet: Each gradient was collected in 28 fractions and analyzed by Western blotting using specific anti-BMP-7 PD (mAb2 from Millipore, Billerica, MA) and GF (500-P198, Peprotech) antibodies ( ).

    Techniques: Variant Assay, Construct, Sequencing