immobilon p polyvinylidene difluoride  (Millipore)


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    Name:
    Immobilon P Polyvinylidene difluoride membranes
    Description:

    Catalog Number:
    p2938
    Price:
    None
    Applications:
    Immobilon-P Polyvinylidene difluoride membrane has been used in western blotting for the transfer of proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
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    Structured Review

    Millipore immobilon p polyvinylidene difluoride
    Effect of CSE on HSA Cys34 free sulfhydryl group as determined by the Ellman assay. HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% (v/v) CSE and then exhaustively dialyzed. The concentration of Cys34 sulfhydryl groups in HSA samples was determined by the Ellman assay at 412 nm as described under Materials and methods . Data are presented as the mean ± SD of three independent measurements. Inset: Effect of CSE on HSA Cys34 free sulfhydryl group as determined by biotin-HPDP binding and Western blot analysis. HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% CSE, exhaustively dialyzed and then labeled at Cys34 with biotin-HPDP as described under Materials and methods . Proteins (10 µg/lane) were separated by SDS-PAGE and biotin-HPDP binding was detected by Western blot analysis using streptavidin-HRP as described under Materials and methods (immunoblot inset). Amido Black staining of the same <t>PVDF</t> membrane showed equal protein loading and transfer (not shown). Immunoblot shown is representative of three independent determinations. Bar-graph inset shows densitometric analysis of biotin-HPDP incorporation. Data are presented as the mean ± SD of three independent determinations.

    https://www.bioz.com/result/immobilon p polyvinylidene difluoride/product/Millipore
    Average 99 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    immobilon p polyvinylidene difluoride - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Red Blood Cells Protect Albumin from Cigarette Smoke-Induced Oxidation"

    Article Title: Red Blood Cells Protect Albumin from Cigarette Smoke-Induced Oxidation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029930

    Effect of CSE on HSA Cys34 free sulfhydryl group as determined by the Ellman assay. HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% (v/v) CSE and then exhaustively dialyzed. The concentration of Cys34 sulfhydryl groups in HSA samples was determined by the Ellman assay at 412 nm as described under Materials and methods . Data are presented as the mean ± SD of three independent measurements. Inset: Effect of CSE on HSA Cys34 free sulfhydryl group as determined by biotin-HPDP binding and Western blot analysis. HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% CSE, exhaustively dialyzed and then labeled at Cys34 with biotin-HPDP as described under Materials and methods . Proteins (10 µg/lane) were separated by SDS-PAGE and biotin-HPDP binding was detected by Western blot analysis using streptavidin-HRP as described under Materials and methods (immunoblot inset). Amido Black staining of the same PVDF membrane showed equal protein loading and transfer (not shown). Immunoblot shown is representative of three independent determinations. Bar-graph inset shows densitometric analysis of biotin-HPDP incorporation. Data are presented as the mean ± SD of three independent determinations.
    Figure Legend Snippet: Effect of CSE on HSA Cys34 free sulfhydryl group as determined by the Ellman assay. HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% (v/v) CSE and then exhaustively dialyzed. The concentration of Cys34 sulfhydryl groups in HSA samples was determined by the Ellman assay at 412 nm as described under Materials and methods . Data are presented as the mean ± SD of three independent measurements. Inset: Effect of CSE on HSA Cys34 free sulfhydryl group as determined by biotin-HPDP binding and Western blot analysis. HSA-SH solutions (60 µM) were treated for 60 min with vehicle (control) or 1%, 4% and 16% CSE, exhaustively dialyzed and then labeled at Cys34 with biotin-HPDP as described under Materials and methods . Proteins (10 µg/lane) were separated by SDS-PAGE and biotin-HPDP binding was detected by Western blot analysis using streptavidin-HRP as described under Materials and methods (immunoblot inset). Amido Black staining of the same PVDF membrane showed equal protein loading and transfer (not shown). Immunoblot shown is representative of three independent determinations. Bar-graph inset shows densitometric analysis of biotin-HPDP incorporation. Data are presented as the mean ± SD of three independent determinations.

    Techniques Used: Concentration Assay, Binding Assay, Western Blot, Labeling, SDS Page, Staining

    Related Articles

    Electrophoresis:

    Article Title: Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae *
    Article Snippet: .. Protein samples incubated with SDS sample buffer (50 m m Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromphenol blue, 1.33% β-mercaptoethanol) were separated on 12% Tris-glycine SDS-polyacrylamide gels by electrophoresis ( ) followed by transfer to Immobilon-P PVDF membranes (Millipore) at 100 V for 1.5 h. Membranes were then blocked overnight in 3% nonfat milk, phosphate-buffered saline (140.7 m m NaCl, 9.3 m m Na2 HPO4 , pH 7.4), 0.1% Tween 20. .. Membranes were then probed with primary antibodies ( ) in 2% nonfat milk, phosphate-buffered saline, 0.1% Tween 20 at the dilutions stated ( ).

    Article Title: The ClpXP ATP-Dependent Protease Regulates Flagellum Synthesis in Salmonella enterica Serovar Typhimurium
    Article Snippet: .. For immunoblot analysis, proteins separated on the SDS-12% polyacrylamide gels were transferred after electrophoresis onto Immobilon-P (Millipore) as previously reported ( ). .. Proteins were reacted with either rabbit anti- S. enterica serovar Typhimurium FliC (1:25,000) or FliA (1:12,500) antiserum, followed by treatment with alkaline phosphatase-conjugated anti-rabbit immunoglobulin G. The enzymatic reactions were performed in the presence of 300 μg of nitroblue tetrazolium (Dojindo, Kumamoto, Japan)/ml and 150 μg of bromochloroindolyl phosphate (aMReSCO, Solon, Ohio)/ml.

    Article Title: Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate
    Article Snippet: .. The gel was soaked in buffer containing 12.5 mM Tris base, 96 mM glycine, and 20% methanol, and the protein was transferred to an Immobilon P (Millipore) membrane by electrophoresis at 100 V for 1 hr in the same buffer. .. The four lanes containing equal amounts of radioactivity were cut into strips and counted in a Beckman scintillation counter.

    Incubation:

    Article Title: Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae *
    Article Snippet: .. Protein samples incubated with SDS sample buffer (50 m m Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromphenol blue, 1.33% β-mercaptoethanol) were separated on 12% Tris-glycine SDS-polyacrylamide gels by electrophoresis ( ) followed by transfer to Immobilon-P PVDF membranes (Millipore) at 100 V for 1.5 h. Membranes were then blocked overnight in 3% nonfat milk, phosphate-buffered saline (140.7 m m NaCl, 9.3 m m Na2 HPO4 , pH 7.4), 0.1% Tween 20. .. Membranes were then probed with primary antibodies ( ) in 2% nonfat milk, phosphate-buffered saline, 0.1% Tween 20 at the dilutions stated ( ).

    Activity Assay:

    Article Title: Characterization of the autophosphorylation property of HflX, a ribosome‐binding GTPase from Escherichia coli
    Article Snippet: .. The Ni+2 ‐nitrilotriacetic acid resin (QIAGEN GmbH, Hilden, Germany), dNTP mix (Fermentas Life Sciences, Waltham, MA, USA), Immobilon‐P (PVDF, polyvinylidene fluoride) membrane (Millipore, Billerica, MA, USA) and [γ32 ‐P] GTP (specific activity 4500 Ci·mmol−1 ) (BRIT, Mumbai, India) were purchased from the indicated sources. .. Cellulose thin layer (100 μm) chromatography plates (polyester sigmacell type 100 cellulose) of dimensions 20 cm × 20 cm, were purchased from Sigma.

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    Millipore millipore pvdf membrane
    Dependence of thermally on-off ratio on the cross-linker amounts in the range from 0.1-2.0 mol% in for <t>PNIPAAm-PVDF</t> <t>Millipore</t> membrane (P=1.4 bar). For all the membranes, the NIPAAm concentration for polymerization solution was 5 wt%. Data was corrected with viscosity and normalized by permeability at 30°C.
    Millipore Pvdf Membrane, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/millipore pvdf membrane/product/Millipore
    Average 99 stars, based on 358 article reviews
    Price from $9.99 to $1999.99
    millipore pvdf membrane - by Bioz Stars, 2020-08
    99/100 stars
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    Dependence of thermally on-off ratio on the cross-linker amounts in the range from 0.1-2.0 mol% in for PNIPAAm-PVDF Millipore membrane (P=1.4 bar). For all the membranes, the NIPAAm concentration for polymerization solution was 5 wt%. Data was corrected with viscosity and normalized by permeability at 30°C.

    Journal: Journal of membrane science

    Article Title: Development of Bench and Full-Scale Temperature and pH Responsive Functionalized PVDF Membranes with Tunable Properties

    doi: 10.1016/j.memsci.2014.01.033

    Figure Lengend Snippet: Dependence of thermally on-off ratio on the cross-linker amounts in the range from 0.1-2.0 mol% in for PNIPAAm-PVDF Millipore membrane (P=1.4 bar). For all the membranes, the NIPAAm concentration for polymerization solution was 5 wt%. Data was corrected with viscosity and normalized by permeability at 30°C.

    Article Snippet: It should be noted that unlike the Millipore PVDF membrane, the functionalization of full-scale membrane was by dip-coating method.

    Techniques: Concentration Assay, Permeability

    ATR-FTIR spectrum of blank PVDF, PNIPAAm functionalized PVDF Millipore membrane and PNIPAAm-FPAA-PVDFHE Sepro membrane.

    Journal: Journal of membrane science

    Article Title: Development of Bench and Full-Scale Temperature and pH Responsive Functionalized PVDF Membranes with Tunable Properties

    doi: 10.1016/j.memsci.2014.01.033

    Figure Lengend Snippet: ATR-FTIR spectrum of blank PVDF, PNIPAAm functionalized PVDF Millipore membrane and PNIPAAm-FPAA-PVDFHE Sepro membrane.

    Article Snippet: It should be noted that unlike the Millipore PVDF membrane, the functionalization of full-scale membrane was by dip-coating method.

    Techniques:

    Dynamic and Reversible flux response versus ramp change in feed temperature above LCST and below LCST through PNIPAAm-PVDF Millipore membrane at P = 1.4 bar. The inset is the experimental temperature step change approximation. For the polymerization, the NIPAAm concentration was 5 wt%, cross-linker was 0.1mol%.

    Journal: Journal of membrane science

    Article Title: Development of Bench and Full-Scale Temperature and pH Responsive Functionalized PVDF Membranes with Tunable Properties

    doi: 10.1016/j.memsci.2014.01.033

    Figure Lengend Snippet: Dynamic and Reversible flux response versus ramp change in feed temperature above LCST and below LCST through PNIPAAm-PVDF Millipore membrane at P = 1.4 bar. The inset is the experimental temperature step change approximation. For the polymerization, the NIPAAm concentration was 5 wt%, cross-linker was 0.1mol%.

    Article Snippet: It should be noted that unlike the Millipore PVDF membrane, the functionalization of full-scale membrane was by dip-coating method.

    Techniques: Concentration Assay

    The effects of temperature and monomer concentration on dextran rejection with PNIPAAm-PVDF Millipore membrane (5mol% cross-linker) (Mw=2,000,000 g/mol; Stokes radius r s = 26.1nm, calculated from r s =0.27·M w 0.498 ).

    Journal: Journal of membrane science

    Article Title: Development of Bench and Full-Scale Temperature and pH Responsive Functionalized PVDF Membranes with Tunable Properties

    doi: 10.1016/j.memsci.2014.01.033

    Figure Lengend Snippet: The effects of temperature and monomer concentration on dextran rejection with PNIPAAm-PVDF Millipore membrane (5mol% cross-linker) (Mw=2,000,000 g/mol; Stokes radius r s = 26.1nm, calculated from r s =0.27·M w 0.498 ).

    Article Snippet: It should be noted that unlike the Millipore PVDF membrane, the functionalization of full-scale membrane was by dip-coating method.

    Techniques: Concentration Assay

    SEM images of blank PVDF (A) and PNIPAAm-PVDF Millipore membranes (B: 25°C(below LCST); C: 40 °C (above LCST))

    Journal: Journal of membrane science

    Article Title: Development of Bench and Full-Scale Temperature and pH Responsive Functionalized PVDF Membranes with Tunable Properties

    doi: 10.1016/j.memsci.2014.01.033

    Figure Lengend Snippet: SEM images of blank PVDF (A) and PNIPAAm-PVDF Millipore membranes (B: 25°C(below LCST); C: 40 °C (above LCST))

    Article Snippet: It should be noted that unlike the Millipore PVDF membrane, the functionalization of full-scale membrane was by dip-coating method.

    Techniques:

    Effect of monomer (NIPAAm) concentration on water flux at 1.4 bar and calculated effective pore size for PNIPAAm-PVDF Millipore membrane. cross-linker concentration = 1mol%.

    Journal: Journal of membrane science

    Article Title: Development of Bench and Full-Scale Temperature and pH Responsive Functionalized PVDF Membranes with Tunable Properties

    doi: 10.1016/j.memsci.2014.01.033

    Figure Lengend Snippet: Effect of monomer (NIPAAm) concentration on water flux at 1.4 bar and calculated effective pore size for PNIPAAm-PVDF Millipore membrane. cross-linker concentration = 1mol%.

    Article Snippet: It should be noted that unlike the Millipore PVDF membrane, the functionalization of full-scale membrane was by dip-coating method.

    Techniques: Concentration Assay

    p27 BBP/eIF6 is present in all cell lines, and in a cytoskeletal associated pool. (Left) 30 μg of total protein extracts from established cell lines and primary cultures were run on 12% acrylamide gels, transferred on immobilon-P membranes and blotted with the p27 BBP/eIF6 antiserum, followed by ECL detection. The arrow points at the p27 BBP/eIF6 band. The cell lines were mouse NIH/3T3 fibroblasts, human A431 epidermoid carcinoma, human HeLa epitheloid carcinoma, human pancreatic carcinoma FG2, human Jurkat T cells, transformed human keratinocytes HaCaT, human insulinoma cells Rin2A, and human neuroblastoma SK-N-MC. Primary cultures were mouse resting splenocytes and human fibroblasts. (Right) p27 BBP/eIF6 in Xenopus oocytes extracted either in Laemmli buffer (total), or according to the nuclear matrix/intermediate filaments procedure of He et al. (1990, see Materials and Methods). The lane marked soluble contains cytosolic proteins extracted with an isotonic Triton X-100 buffer. The lane marked insoluble contains nuclear matrix/intermediate filament-associated proteins left after sequential extraction with ammonium sulphate, nuclease, and high salt.

    Journal: The Journal of Cell Biology

    Article Title: The ?4 Integrin Interactor p27BBP/eIF6 Is an Essential Nuclear Matrix Protein Involved in 60S Ribosomal Subunit Assembly

    doi:

    Figure Lengend Snippet: p27 BBP/eIF6 is present in all cell lines, and in a cytoskeletal associated pool. (Left) 30 μg of total protein extracts from established cell lines and primary cultures were run on 12% acrylamide gels, transferred on immobilon-P membranes and blotted with the p27 BBP/eIF6 antiserum, followed by ECL detection. The arrow points at the p27 BBP/eIF6 band. The cell lines were mouse NIH/3T3 fibroblasts, human A431 epidermoid carcinoma, human HeLa epitheloid carcinoma, human pancreatic carcinoma FG2, human Jurkat T cells, transformed human keratinocytes HaCaT, human insulinoma cells Rin2A, and human neuroblastoma SK-N-MC. Primary cultures were mouse resting splenocytes and human fibroblasts. (Right) p27 BBP/eIF6 in Xenopus oocytes extracted either in Laemmli buffer (total), or according to the nuclear matrix/intermediate filaments procedure of He et al. (1990, see Materials and Methods). The lane marked soluble contains cytosolic proteins extracted with an isotonic Triton X-100 buffer. The lane marked insoluble contains nuclear matrix/intermediate filament-associated proteins left after sequential extraction with ammonium sulphate, nuclease, and high salt.

    Article Snippet: Western Blot Analysis All samples were denatured before loading in Laemmli buffer ( ) and run on denaturing 12% SDS–acrylamide gel, transferred to Immobilon P membranes ( Millipore Corp. ), and blotted with the rabbit p27BBP/eIF6 antiserum at 1:1,000 dilution as previously described ( ).

    Techniques: Transformation Assay

    p27 BBP/eIF6 cosediments with 60S and 80S ribosomal subunits. Fractions collected after the sucrose gradient centrifugation (top) of extracts from strain ySP664 ( iih1 Δ GAL-Hsp27 BBP/eIF6 ), logarithmically growing in galactose-containing medium, were precipitated by TCA, and equal amounts of protein extracts were run on 12% acrylamide gels, transferred on immobilon-P membranes and probed with the anti–p27 BBP/eIF6 antiserum, followed by the ECL detection method (bottom). Note that p27 BBP/eIF6 (arrow) is highly enriched in the free 60S and 80S fractions, but clearly absent from the polysome fraction. The high molecular weight band in lane 1 is a nonspecific product recognized by secondary antibodies.

    Journal: The Journal of Cell Biology

    Article Title: The ?4 Integrin Interactor p27BBP/eIF6 Is an Essential Nuclear Matrix Protein Involved in 60S Ribosomal Subunit Assembly

    doi:

    Figure Lengend Snippet: p27 BBP/eIF6 cosediments with 60S and 80S ribosomal subunits. Fractions collected after the sucrose gradient centrifugation (top) of extracts from strain ySP664 ( iih1 Δ GAL-Hsp27 BBP/eIF6 ), logarithmically growing in galactose-containing medium, were precipitated by TCA, and equal amounts of protein extracts were run on 12% acrylamide gels, transferred on immobilon-P membranes and probed with the anti–p27 BBP/eIF6 antiserum, followed by the ECL detection method (bottom). Note that p27 BBP/eIF6 (arrow) is highly enriched in the free 60S and 80S fractions, but clearly absent from the polysome fraction. The high molecular weight band in lane 1 is a nonspecific product recognized by secondary antibodies.

    Article Snippet: Western Blot Analysis All samples were denatured before loading in Laemmli buffer ( ) and run on denaturing 12% SDS–acrylamide gel, transferred to Immobilon P membranes ( Millipore Corp. ), and blotted with the rabbit p27BBP/eIF6 antiserum at 1:1,000 dilution as previously described ( ).

    Techniques: Gradient Centrifugation, Molecular Weight

    Western blot analysis of nuclear extracts from macrophages of pIpC-treated PPARγ-MXCre + mice and similarly treated PPARγ-MXCre − mice. A total of 10 μg of protein from nuclear extracts of macrophages and 10 μg of total protein from Hepa-1 cells transfected with an expression vector for PPARγ1 (pSG5-PPARγ cDNA) were subjected to electrophoresis on a 4 to 15% Tris-HCl gradient gel (Bio-Rad), transferred to Immobilon-P membranes (Millipore), and probed as recommended by the manufacturer with anti-PPARγ antibodies (Santa Cruz Biotechnologies) specific for the N terminus (E-8, PPARγ antibody) (A) and the C terminus (H-100, PPARγ antibody) (B) of the PPARγ protein. Detection of immunoreactive proteins was done by using an enhanced chemiluminescence blot detection system (Amersham).

    Journal: Molecular and Cellular Biology

    Article Title: Conditional Disruption of the Peroxisome Proliferator-Activated Receptor ? Gene in Mice Results in Lowered Expression of ABCA1, ABCG1, and apoE in Macrophages and Reduced Cholesterol Efflux

    doi: 10.1128/MCB.22.8.2607-2619.2002

    Figure Lengend Snippet: Western blot analysis of nuclear extracts from macrophages of pIpC-treated PPARγ-MXCre + mice and similarly treated PPARγ-MXCre − mice. A total of 10 μg of protein from nuclear extracts of macrophages and 10 μg of total protein from Hepa-1 cells transfected with an expression vector for PPARγ1 (pSG5-PPARγ cDNA) were subjected to electrophoresis on a 4 to 15% Tris-HCl gradient gel (Bio-Rad), transferred to Immobilon-P membranes (Millipore), and probed as recommended by the manufacturer with anti-PPARγ antibodies (Santa Cruz Biotechnologies) specific for the N terminus (E-8, PPARγ antibody) (A) and the C terminus (H-100, PPARγ antibody) (B) of the PPARγ protein. Detection of immunoreactive proteins was done by using an enhanced chemiluminescence blot detection system (Amersham).

    Article Snippet: Then, 10 μg of total protein from Hepa-1 cells and 10 μg of protein from nuclear extracts of macrophages were subjected to electrophoresis on a 4 to 15% Tris-HCl gradient gel (Bio-Rad, Hercules, Calif.), transferred to Immobilon-P membranes (Millipore, Bedford, Mass.), and probed according to the manufacturer's recommendations with anti-PPARγ antibodies (E-8 and H-100; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) as indicated.

    Techniques: Western Blot, Mouse Assay, Transfection, Expressing, Plasmid Preparation, Electrophoresis

    GSK-3β binds to D1 subunits and preferentially phosphorylates cyclin D1 in complexes with CDK4. ( A ) Sf9 cells infected with baculoviruses encoding cyclin D1 or cyclin D1 plus CDK4 with or without wild-type GSK-3β as indicated were metabolically labeled with [ 32 P]orthophosphate. Lysates were subjected to precipitation with NRS (lane 1 ), antibody to cyclin D1 (lanes 2 , 3 ), or antiserum specific for the carboxyl terminus of CDK4 (lanes 4 , 5 ) as indicated. Phosphorylated proteins were resolved on denaturing polyacrylamide gels and transferred to a nitrocellulose membrane. Following autoradiography, the membrane was blotted with the antibody to cyclin D1 ( bottom ) and sites of antibody binding were visualized by enhanced chemiluminescence. ( B ) Sf9 cells infected with baculoviruses encoding cyclin D1 and CDK4 together with wild-type (wt) or kinase-defective (kd) GSK-3β were labeled with [ 32 P]orthophosphate. Lysates were subjected to precipitation with NRS or anti-D1 as indicated, and phosphorylated proteins were resolved on a denaturing polyacrylamide gel followed by transfer to Immobilon-P membrane (autoradiographic exposure time 12 hr; top ). Following autoradiography, the membrane was blotted with the antibody to cyclin D1 as in A . The relative ratio of 32 P-labeled cyclin D1 versus total cyclin D1 in each lysate (densitometric scanning) is indicated between the two autoradiographs. ( C ) Sf9 lysates infected with baculoviruses encoding the proteins indicated were precipitated with the indicated antibodies and blotted with a monoclonal antibody to GSK-3β. Sites of antibody binding were visualized by enhanced chemiluminesence.

    Journal: Genes & Development

    Article Title: Glycogen synthase kinase-3? regulates cyclin D1 proteolysis and subcellular localization

    doi:

    Figure Lengend Snippet: GSK-3β binds to D1 subunits and preferentially phosphorylates cyclin D1 in complexes with CDK4. ( A ) Sf9 cells infected with baculoviruses encoding cyclin D1 or cyclin D1 plus CDK4 with or without wild-type GSK-3β as indicated were metabolically labeled with [ 32 P]orthophosphate. Lysates were subjected to precipitation with NRS (lane 1 ), antibody to cyclin D1 (lanes 2 , 3 ), or antiserum specific for the carboxyl terminus of CDK4 (lanes 4 , 5 ) as indicated. Phosphorylated proteins were resolved on denaturing polyacrylamide gels and transferred to a nitrocellulose membrane. Following autoradiography, the membrane was blotted with the antibody to cyclin D1 ( bottom ) and sites of antibody binding were visualized by enhanced chemiluminescence. ( B ) Sf9 cells infected with baculoviruses encoding cyclin D1 and CDK4 together with wild-type (wt) or kinase-defective (kd) GSK-3β were labeled with [ 32 P]orthophosphate. Lysates were subjected to precipitation with NRS or anti-D1 as indicated, and phosphorylated proteins were resolved on a denaturing polyacrylamide gel followed by transfer to Immobilon-P membrane (autoradiographic exposure time 12 hr; top ). Following autoradiography, the membrane was blotted with the antibody to cyclin D1 as in A . The relative ratio of 32 P-labeled cyclin D1 versus total cyclin D1 in each lysate (densitometric scanning) is indicated between the two autoradiographs. ( C ) Sf9 lysates infected with baculoviruses encoding the proteins indicated were precipitated with the indicated antibodies and blotted with a monoclonal antibody to GSK-3β. Sites of antibody binding were visualized by enhanced chemiluminesence.

    Article Snippet: Phosphorylated proteins were resolved on denaturing polyacrylamide gels, transferred to Immobilon-P membranes (Millipore), and visualized by autoradiographic exposure.

    Techniques: Infection, Metabolic Labelling, Labeling, Autoradiography, Binding Assay

    GSK-3β phosphorylates cyclin D1 on Thr-286. ( A ) Cyclin D1 (odd lanes) or cyclin D1-(T286A) (even lanes) immunoprecipitated from Sf9 cells infected with baculoviruses encoding D1 and CDK4 were mixed with recombinant GSK-3β (lanes 1 , 2 ), ERK2 (lanes 3 , 4 ), SAPK (lanes 5 , 6 ), or cyclin E–CDK2 (lanes 7 , 8 ) plus [γ- 32 P]ATP. After incubation at 30°C for 30 min, phosphorylated proteins were separated on a denaturing polyacrylamide gel, transferred to an Immobilon-P membrane, and visualized by autoradiography. The position of phosphorylated cyclin D1 is indicated. ( B ) Membrane slices containing cyclin D1 phosphorylated by GSK-3β in vitro ( left ), phosphorylated by endogenous Sf9 kinases ( middle ), or by a mixture of the two ( right ).

    Journal: Genes & Development

    Article Title: Glycogen synthase kinase-3? regulates cyclin D1 proteolysis and subcellular localization

    doi:

    Figure Lengend Snippet: GSK-3β phosphorylates cyclin D1 on Thr-286. ( A ) Cyclin D1 (odd lanes) or cyclin D1-(T286A) (even lanes) immunoprecipitated from Sf9 cells infected with baculoviruses encoding D1 and CDK4 were mixed with recombinant GSK-3β (lanes 1 , 2 ), ERK2 (lanes 3 , 4 ), SAPK (lanes 5 , 6 ), or cyclin E–CDK2 (lanes 7 , 8 ) plus [γ- 32 P]ATP. After incubation at 30°C for 30 min, phosphorylated proteins were separated on a denaturing polyacrylamide gel, transferred to an Immobilon-P membrane, and visualized by autoradiography. The position of phosphorylated cyclin D1 is indicated. ( B ) Membrane slices containing cyclin D1 phosphorylated by GSK-3β in vitro ( left ), phosphorylated by endogenous Sf9 kinases ( middle ), or by a mixture of the two ( right ).

    Article Snippet: Phosphorylated proteins were resolved on denaturing polyacrylamide gels, transferred to Immobilon-P membranes (Millipore), and visualized by autoradiographic exposure.

    Techniques: Immunoprecipitation, Infection, Recombinant, Incubation, Autoradiography, In Vitro