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Bio-Rad recombinant proteins
Recombinant Proteins, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 272 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 272 article reviews
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recombinant proteins - by Bioz Stars, 2020-11
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Article Title: Phosphorylation of TGB1 by protein kinase CK2 promotes barley stripe mosaic virus movement in monocots and dicots
Article Snippet: Recombinant proteins were extracted from the bacteria and purified by Ni-affinity chromatography according to the manufacturer’s instructions (Bio-Rad) and evaluated by 12.5% SDS-PAGE.

Article Title: A role for trypanosomatid aldo-keto reductases in methylglyoxal, prostaglandin and isoprostane metabolism
Article Snippet: Recombinant proteins were loaded onto a Superdex 75 10/300 or Superose 12 10/300 column, equilibrated in 25 mM HEPES, 150 mM NaCl, pH 7.33 and the elution volume of the recombinant proteins was compared with that of known protein standards (Bio-Rad).

Article Title: Seroprevalence of Turkey Coronavirus in North American Turkeys Determined by a Newly Developed Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigen ▿
Article Snippet: Purified recombinant proteins were visualized using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie blue G-250 (Bio-Rad, Hercules, CA).

Article Title: Cloning, Expression, and Immunological Evaluation of Two Putative Secreted Serine Protease Antigens of Mycobacterium tuberculosis
Article Snippet: M. tuberculosis (strain H37Rv) total lysate or CFP (2.5 μg each) as well as 50 ng of the indicated recombinant proteins were separated by electrophoresis on sodium dodecyl sulfate (SDS)–15% polyacrylamide gels and transferred to nitrocellulose with a semidry transfer apparatus (Bio-Rad).

Article Title: Conformational IgE Epitope Mapping of Der p 2 and the Evaluations of Two Candidate Hypoallergens for Immunotherapy
Article Snippet: Immuno dot-blot One microgram of crude allergen extract or recombinant proteins were dotted on nitrocellulose membranes (BIO-RAD Laboratories, USA), air dried and blocked with PBS-0.1% Tween-20.

Article Title: Identification of novel binding sites for heparin in receptor protein-tyrosine phosphatase (RPTPσ): Implications for proteoglycan signaling
Article Snippet: Secreted recombinant proteins were purified by immobilized metal-affinity chromatography for AP fusion proteins (Profinity IMAC nickel-charged resin, Bio-Rad) and by Protein G-Sepharose 4 Fast Flow for Fc fusion proteins (GE Healthcare).

Article Title: Plasmodium vivax: comparison of immunogenicity among proteins expressed in the cell-free systems of Escherichia coli and wheat germ by suspension array assays
Article Snippet: Covalent coupling of recombinant proteins to beads BioPlex carboxylated beads (Bio-Rad) were covalently coated with the different recombinant proteins following the manufacturer's instructions (BioPlex Amine Coupling Kit).

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  • 85
    Bio-Rad gst purified recombinant proteins
    Expression and purification of <t>rOPN-GST</t> fusion protein. rOPN was produced as a fusion protein with GST in E. coli BL21. (Left panel) Purified rOPN-GST was separated by 12% SDS-PAGE and stained with Coomassie brillant blue. (Right panel) The separated fusion proteins were transferred onto nitrocellulose membranes and then incubated with an <t>anti-OPN</t> monoclonal antibody.
    Gst Purified Recombinant Proteins, supplied by Bio-Rad, 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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    88
    Bio-Rad recombinant proteins atxβ
    Schematic representation of “classical ATX” (ATXα, β, γ) and “novel ATX” (ATXδ, ε). The amino acid numbers corresponding to human <t>ATXβ</t> and ATXδ are shown, respectively. “Novel ATX” contains a 4-amino acid deletion in the L2 linker.
    Recombinant Proteins Atxβ, supplied by Bio-Rad, 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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    91
    Bio-Rad nhfs
    Passage through the G1/S restriction point and initiation of DNA synthesis are necessary for CDK2-induced PCNA monoubiquitination. (A) <t>NHFs</t> were infected with the indicated adenoviral vectors for 24 h, and cell lysates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (B) Replicate cultures of NHFs and shp53-NHF cells were <t>transfected</t> with siRAD18 or control nontargeting RNA. The resulting cultures were infected with the indicated viruses. One plate of each replicate culture was analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. The other plate of each replicate pair was pulse labeled with BrdU for 1 h. The resulting cultures were costained with propidium iodide and BrdU and then analyzed for cell cycle distribution as shown in C. The white dotted line serves to separate the NHF and shp53-NHF data for easier comparison of protein levels between the two cell lines. (C) Graphs summarizing the distribution of cells between different cell cycle phases for each experimental condition. (D) Swiss 3T3-XSN and 3T3-E6 cells were transfected with siRAD18 or with nontargeting siRNA and then treated with the indicated concentrations of MK-1775 for 5 h. Cell lysates were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (E) Quiescent (G0) cultures of Swiss 3T3-XSN and 3T3-E6 cells were stimulated with 10% serum. At 8 and 16 h after serum stimulation (time points corresponding to mid-G1 and S phase, respectively), cells were treated with MK-1775 for 5 h before harvest, SDS-PAGE, and immunoblot analysis of the indicated proteins. (F) NHFs were infected with AdCyclin E (or empty vector for control) for 24 h. The resulting cultures were treated with roscovitine for 2 h before harvest for SDS-PAGE and immunoblot analysis. (G) NHFs were transfected with siCDC6, siCDC7, or nontargeting RNA duplexes. The resulting cultures were infected with AdCyclin E (or empty adenoviral vector). 48 h later, cells were harvested for SDS-PAGE and immunoblotting with the indicated antibodies.
    Nhfs, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Bio-Rad mouse pai 1
    <t>PAI-1</t> expression in mice and human subjects. A and B , plasma levels of PAI-1 in mice as measured by ELISA. Berk-hemi , hemizygous Berkeley mice; Berk-SS , Berkeley SS mice. C , plasma PlGF levels in mice by ELISA. The respective strain is indicated at the bottom of the figure. D , immunohistochemistry for PAI-1 expression in C57BL/6 (normal) and Berkeley SS mouse lungs (shown in brown ; nuclei are stained red ). The top panel shows: (i) antibody control and PAI-1 expression in bronchial epithelial cells of normal and (ii) Berkeley SS mice; the bottom panel shows corresponding staining for PAI-1 in their lung parenchyma. Arrows indicate alveolar macrophages, and arrowheads indicate bronchial epithelial layer. E , the levels of PAI-1 were quantified in BAL of indicated strains of mice by ELISA. F , qRT-PCR analysis of PAI-1 mRNA in MNC from SCD subjects ( n = 9) and normal controls ( n = 9). RQ , relative quantification. G and H , HPMVEC were treated with human recombinant PlGF (250 ng/ml) for the indicated time periods. G , total RNA was subjected to RPA for the expression of indicated genes. H , the culture supernatants were assayed for PAI-1 by ELISA. RPA data are representative of three independent experiments. GAPDH , glyceraldehyde-3-phosphate dehydrogenase. Where indicated, the vertical lines show repositioned gel lanes. *, p
    Mouse Pai 1, supplied by Bio-Rad, 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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    Image Search Results


    Expression and purification of rOPN-GST fusion protein. rOPN was produced as a fusion protein with GST in E. coli BL21. (Left panel) Purified rOPN-GST was separated by 12% SDS-PAGE and stained with Coomassie brillant blue. (Right panel) The separated fusion proteins were transferred onto nitrocellulose membranes and then incubated with an anti-OPN monoclonal antibody.

    Journal: Oncology Letters

    Article Title: Osteopontin is a tumor autoantigen in prostate cancer patients

    doi: 10.3892/ol.2010.211

    Figure Lengend Snippet: Expression and purification of rOPN-GST fusion protein. rOPN was produced as a fusion protein with GST in E. coli BL21. (Left panel) Purified rOPN-GST was separated by 12% SDS-PAGE and stained with Coomassie brillant blue. (Right panel) The separated fusion proteins were transferred onto nitrocellulose membranes and then incubated with an anti-OPN monoclonal antibody.

    Article Snippet: For immunoblot assays, 50 μg of GST-OPN and GST-purified recombinant proteins were subjected to electrophoresis via a 12% SDS-polyacrylamide gel and then transferred onto nitrocellulose membranes using the mini-PROTEAN 3 (Bio-Rad) system.

    Techniques: Expressing, Purification, Produced, SDS Page, Staining, Incubation

    PCa patients present higher reactivity levels of anti-OPN antibodies as compared to BPH and the HD controls. A representative immunoblot of plasma samples assayed for their reactivity to recombinant OPN is shown. Plasma samples from 29 biopsy-proven PCa, 18 BPH and 30 HD were tested. The plasma samples were tested in 1:200 dilution and the antigen-antibody complexes were probed with AP-conjugated anti-human IgG antibody. The assays were performed simultaneously using the same batch of recombinant OPN. The representative plasma samples tested are numbered above each panel. The detection of immunoreactive bands was performed using NBT/BCIP as AP substrates. The two major reactive rOPN-GST (lane OG) protein fragments and GST alone (lane G) were used as a negative control and their molecular weight sizes are indicated by the arrows. None of the tested pre-adsorbed plasma samples were reactive for GST.

    Journal: Oncology Letters

    Article Title: Osteopontin is a tumor autoantigen in prostate cancer patients

    doi: 10.3892/ol.2010.211

    Figure Lengend Snippet: PCa patients present higher reactivity levels of anti-OPN antibodies as compared to BPH and the HD controls. A representative immunoblot of plasma samples assayed for their reactivity to recombinant OPN is shown. Plasma samples from 29 biopsy-proven PCa, 18 BPH and 30 HD were tested. The plasma samples were tested in 1:200 dilution and the antigen-antibody complexes were probed with AP-conjugated anti-human IgG antibody. The assays were performed simultaneously using the same batch of recombinant OPN. The representative plasma samples tested are numbered above each panel. The detection of immunoreactive bands was performed using NBT/BCIP as AP substrates. The two major reactive rOPN-GST (lane OG) protein fragments and GST alone (lane G) were used as a negative control and their molecular weight sizes are indicated by the arrows. None of the tested pre-adsorbed plasma samples were reactive for GST.

    Article Snippet: For immunoblot assays, 50 μg of GST-OPN and GST-purified recombinant proteins were subjected to electrophoresis via a 12% SDS-polyacrylamide gel and then transferred onto nitrocellulose membranes using the mini-PROTEAN 3 (Bio-Rad) system.

    Techniques: Recombinant, Negative Control, Molecular Weight

    Schematic representation of “classical ATX” (ATXα, β, γ) and “novel ATX” (ATXδ, ε). The amino acid numbers corresponding to human ATXβ and ATXδ are shown, respectively. “Novel ATX” contains a 4-amino acid deletion in the L2 linker.

    Journal: PLoS ONE

    Article Title: A New Enzyme Immunoassay for the Quantitative Determination of Classical Autotaxins (ATXα, ATXβ, and ATXγ) and Novel Autotaxins (ATXδ and ATXε)

    doi: 10.1371/journal.pone.0130074

    Figure Lengend Snippet: Schematic representation of “classical ATX” (ATXα, β, γ) and “novel ATX” (ATXδ, ε). The amino acid numbers corresponding to human ATXβ and ATXδ are shown, respectively. “Novel ATX” contains a 4-amino acid deletion in the L2 linker.

    Article Snippet: The purified recombinant proteins ATXβ and ATXδ and serum samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to a polyvinylidene fluoride membrane using a semi-dry blotter (Transblot SD Cell, Bio-Rad).

    Techniques:

    Passage through the G1/S restriction point and initiation of DNA synthesis are necessary for CDK2-induced PCNA monoubiquitination. (A) NHFs were infected with the indicated adenoviral vectors for 24 h, and cell lysates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (B) Replicate cultures of NHFs and shp53-NHF cells were transfected with siRAD18 or control nontargeting RNA. The resulting cultures were infected with the indicated viruses. One plate of each replicate culture was analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. The other plate of each replicate pair was pulse labeled with BrdU for 1 h. The resulting cultures were costained with propidium iodide and BrdU and then analyzed for cell cycle distribution as shown in C. The white dotted line serves to separate the NHF and shp53-NHF data for easier comparison of protein levels between the two cell lines. (C) Graphs summarizing the distribution of cells between different cell cycle phases for each experimental condition. (D) Swiss 3T3-XSN and 3T3-E6 cells were transfected with siRAD18 or with nontargeting siRNA and then treated with the indicated concentrations of MK-1775 for 5 h. Cell lysates were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (E) Quiescent (G0) cultures of Swiss 3T3-XSN and 3T3-E6 cells were stimulated with 10% serum. At 8 and 16 h after serum stimulation (time points corresponding to mid-G1 and S phase, respectively), cells were treated with MK-1775 for 5 h before harvest, SDS-PAGE, and immunoblot analysis of the indicated proteins. (F) NHFs were infected with AdCyclin E (or empty vector for control) for 24 h. The resulting cultures were treated with roscovitine for 2 h before harvest for SDS-PAGE and immunoblot analysis. (G) NHFs were transfected with siCDC6, siCDC7, or nontargeting RNA duplexes. The resulting cultures were infected with AdCyclin E (or empty adenoviral vector). 48 h later, cells were harvested for SDS-PAGE and immunoblotting with the indicated antibodies.

    Journal: The Journal of Cell Biology

    Article Title: DNA repair factor RAD18 and DNA polymerase Polκ confer tolerance of oncogenic DNA replication stress

    doi: 10.1083/jcb.201702006

    Figure Lengend Snippet: Passage through the G1/S restriction point and initiation of DNA synthesis are necessary for CDK2-induced PCNA monoubiquitination. (A) NHFs were infected with the indicated adenoviral vectors for 24 h, and cell lysates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (B) Replicate cultures of NHFs and shp53-NHF cells were transfected with siRAD18 or control nontargeting RNA. The resulting cultures were infected with the indicated viruses. One plate of each replicate culture was analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. The other plate of each replicate pair was pulse labeled with BrdU for 1 h. The resulting cultures were costained with propidium iodide and BrdU and then analyzed for cell cycle distribution as shown in C. The white dotted line serves to separate the NHF and shp53-NHF data for easier comparison of protein levels between the two cell lines. (C) Graphs summarizing the distribution of cells between different cell cycle phases for each experimental condition. (D) Swiss 3T3-XSN and 3T3-E6 cells were transfected with siRAD18 or with nontargeting siRNA and then treated with the indicated concentrations of MK-1775 for 5 h. Cell lysates were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (E) Quiescent (G0) cultures of Swiss 3T3-XSN and 3T3-E6 cells were stimulated with 10% serum. At 8 and 16 h after serum stimulation (time points corresponding to mid-G1 and S phase, respectively), cells were treated with MK-1775 for 5 h before harvest, SDS-PAGE, and immunoblot analysis of the indicated proteins. (F) NHFs were infected with AdCyclin E (or empty vector for control) for 24 h. The resulting cultures were treated with roscovitine for 2 h before harvest for SDS-PAGE and immunoblot analysis. (G) NHFs were transfected with siCDC6, siCDC7, or nontargeting RNA duplexes. The resulting cultures were infected with AdCyclin E (or empty adenoviral vector). 48 h later, cells were harvested for SDS-PAGE and immunoblotting with the indicated antibodies.

    Article Snippet: Cells were then trypsinized and resuspended in 1 ml of OptiMEM and added directly into the siRNA/OptiMEM/Lipofectamine solution to give a plating density of 50%, and then they were incubated for 48 h. For NHFs and mouse fibroblasts, siRNAs were transfected using electroporation with a GenePulser Xcell (Bio-Rad Laboratories).

    Techniques: DNA Synthesis, Infection, SDS Page, Transfection, Labeling, Plasmid Preparation

    CDK2-induced PCNA monoubiquitination is p95/NBS1-independent but requires MUS81. (A) NHFs were transfected with siNBS1, siRPA32, siRNF8, siCHK1, or control nontargeting siRNA. The resulting cultures were infected with AdCyclin E or control adenovirus, and 48 h later, cells were harvested for SDS-PAGE and immunoblot analysis. (B) Replicate cultures of NHFs were infected with AdCyclin E for 24 h, treated with MK-1775 for 5 h, or left untreated for controls. The resulting cells were fixed, stained with anti-RPA 32 and DAPI, and analyzed by confocal microscopy. The images represent representative fields of RPA- and DAPI-stained nuclei. At least 100 cells were scored for each experimental condition. Bar, 10 µm. (C) NHFs were transfected with siRAD18 or nontargeting siRNA and then treated with MK-1775 for 5 h. Cells were harvested and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (D) NHFs were infected with the indicated adenoviruses for 48 h. Nuclei from the resulting cultures were analyzed using alkaline and neutral comet assays. 50 tail moments were measured for each experimental condition. To determine the statistical significance of the differences in single-strand break (SSB) and DSB levels, we performed ANOVA between groups followed by Tukey’s multiple comparison of means test. Results of the Tukey test indicated significant differences between AdCon and AdCyclin E–overexpressing cells (P

    Journal: The Journal of Cell Biology

    Article Title: DNA repair factor RAD18 and DNA polymerase Polκ confer tolerance of oncogenic DNA replication stress

    doi: 10.1083/jcb.201702006

    Figure Lengend Snippet: CDK2-induced PCNA monoubiquitination is p95/NBS1-independent but requires MUS81. (A) NHFs were transfected with siNBS1, siRPA32, siRNF8, siCHK1, or control nontargeting siRNA. The resulting cultures were infected with AdCyclin E or control adenovirus, and 48 h later, cells were harvested for SDS-PAGE and immunoblot analysis. (B) Replicate cultures of NHFs were infected with AdCyclin E for 24 h, treated with MK-1775 for 5 h, or left untreated for controls. The resulting cells were fixed, stained with anti-RPA 32 and DAPI, and analyzed by confocal microscopy. The images represent representative fields of RPA- and DAPI-stained nuclei. At least 100 cells were scored for each experimental condition. Bar, 10 µm. (C) NHFs were transfected with siRAD18 or nontargeting siRNA and then treated with MK-1775 for 5 h. Cells were harvested and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (D) NHFs were infected with the indicated adenoviruses for 48 h. Nuclei from the resulting cultures were analyzed using alkaline and neutral comet assays. 50 tail moments were measured for each experimental condition. To determine the statistical significance of the differences in single-strand break (SSB) and DSB levels, we performed ANOVA between groups followed by Tukey’s multiple comparison of means test. Results of the Tukey test indicated significant differences between AdCon and AdCyclin E–overexpressing cells (P

    Article Snippet: Cells were then trypsinized and resuspended in 1 ml of OptiMEM and added directly into the siRNA/OptiMEM/Lipofectamine solution to give a plating density of 50%, and then they were incubated for 48 h. For NHFs and mouse fibroblasts, siRNAs were transfected using electroporation with a GenePulser Xcell (Bio-Rad Laboratories).

    Techniques: Transfection, Infection, SDS Page, Staining, Recombinase Polymerase Amplification, Confocal Microscopy

    Oncogenic stimuli promote RAD18-mediated PCNA monoubiquitination. (A) Adenoviral vectors were used to express c-MYC, Ha-RAS V12 , CDT1, and Cyclin E in cultured NHF. After 48 h, cell lysates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (B) Immunoblot showing relative Cyclin E protein levels in AdCyclin E–infected NHFs and various cancer cell lines. Soluble extracts from the indicated cells were normalized for protein concentration and then analyzed by SDS-PAGE and immunoblotting using antibodies against Cyclin E and GAPDH (for loading controls). (C) IP–kinase assay showing relative CDK2 activities in RAS-transformed cells (BJ-RAS) and isogenic parental fibroblasts (BJ). Also shown are CDK2 activities in control, MK-1775–treated, and Cyclin E–overexpressing NHFs and in H1299 lung adenocarcinoma cells. (D) Immunoblot showing the effects of 24 h roscovitine treatment on PCNA monoubiquitination in BJ and BJ-RAS cells. (E) Control and RAD18-depleted NHF cells were treated for 0–7 h with MK-1775. At different times, cell lysates were analyzed for expression of the indicated DNA damage markers using SDS-PAGE and immunoblotting. (F) Rad18 +/+ and Rad18 −/− MEFs were treated with MK-1775 for 7 h. Lysates from the resulting cells were analyzed by SDS-PAGE and immunoblotted with indicated antibodies. (G) U2OS cells were coinfected with AdCFP-RAD18 and AdCyclin E (or with control adenovirus) for 24 h. Some cultures were UV irradiated (20 J/m 2 ), and 2 h later, CFP-RAD18 subcellular distribution was analyzed by confocal microscopy. Bar, 10 µm. (H) NHFs were transfected with siRAD18 or with nontargeting control siRNA. Transfected cells were infected with adenoviruses encoding siRNA-resistant RAD18, Cyclin E, or with an “empty” virus for control. Lysates from the resulting cells were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies.

    Journal: The Journal of Cell Biology

    Article Title: DNA repair factor RAD18 and DNA polymerase Polκ confer tolerance of oncogenic DNA replication stress

    doi: 10.1083/jcb.201702006

    Figure Lengend Snippet: Oncogenic stimuli promote RAD18-mediated PCNA monoubiquitination. (A) Adenoviral vectors were used to express c-MYC, Ha-RAS V12 , CDT1, and Cyclin E in cultured NHF. After 48 h, cell lysates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. (B) Immunoblot showing relative Cyclin E protein levels in AdCyclin E–infected NHFs and various cancer cell lines. Soluble extracts from the indicated cells were normalized for protein concentration and then analyzed by SDS-PAGE and immunoblotting using antibodies against Cyclin E and GAPDH (for loading controls). (C) IP–kinase assay showing relative CDK2 activities in RAS-transformed cells (BJ-RAS) and isogenic parental fibroblasts (BJ). Also shown are CDK2 activities in control, MK-1775–treated, and Cyclin E–overexpressing NHFs and in H1299 lung adenocarcinoma cells. (D) Immunoblot showing the effects of 24 h roscovitine treatment on PCNA monoubiquitination in BJ and BJ-RAS cells. (E) Control and RAD18-depleted NHF cells were treated for 0–7 h with MK-1775. At different times, cell lysates were analyzed for expression of the indicated DNA damage markers using SDS-PAGE and immunoblotting. (F) Rad18 +/+ and Rad18 −/− MEFs were treated with MK-1775 for 7 h. Lysates from the resulting cells were analyzed by SDS-PAGE and immunoblotted with indicated antibodies. (G) U2OS cells were coinfected with AdCFP-RAD18 and AdCyclin E (or with control adenovirus) for 24 h. Some cultures were UV irradiated (20 J/m 2 ), and 2 h later, CFP-RAD18 subcellular distribution was analyzed by confocal microscopy. Bar, 10 µm. (H) NHFs were transfected with siRAD18 or with nontargeting control siRNA. Transfected cells were infected with adenoviruses encoding siRNA-resistant RAD18, Cyclin E, or with an “empty” virus for control. Lysates from the resulting cells were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies.

    Article Snippet: Cells were then trypsinized and resuspended in 1 ml of OptiMEM and added directly into the siRNA/OptiMEM/Lipofectamine solution to give a plating density of 50%, and then they were incubated for 48 h. For NHFs and mouse fibroblasts, siRNAs were transfected using electroporation with a GenePulser Xcell (Bio-Rad Laboratories).

    Techniques: Cell Culture, SDS Page, Infection, Protein Concentration, IP-Kinase Assay, Transformation Assay, Expressing, Irradiation, Confocal Microscopy, Transfection

    PAI-1 expression in mice and human subjects. A and B , plasma levels of PAI-1 in mice as measured by ELISA. Berk-hemi , hemizygous Berkeley mice; Berk-SS , Berkeley SS mice. C , plasma PlGF levels in mice by ELISA. The respective strain is indicated at the bottom of the figure. D , immunohistochemistry for PAI-1 expression in C57BL/6 (normal) and Berkeley SS mouse lungs (shown in brown ; nuclei are stained red ). The top panel shows: (i) antibody control and PAI-1 expression in bronchial epithelial cells of normal and (ii) Berkeley SS mice; the bottom panel shows corresponding staining for PAI-1 in their lung parenchyma. Arrows indicate alveolar macrophages, and arrowheads indicate bronchial epithelial layer. E , the levels of PAI-1 were quantified in BAL of indicated strains of mice by ELISA. F , qRT-PCR analysis of PAI-1 mRNA in MNC from SCD subjects ( n = 9) and normal controls ( n = 9). RQ , relative quantification. G and H , HPMVEC were treated with human recombinant PlGF (250 ng/ml) for the indicated time periods. G , total RNA was subjected to RPA for the expression of indicated genes. H , the culture supernatants were assayed for PAI-1 by ELISA. RPA data are representative of three independent experiments. GAPDH , glyceraldehyde-3-phosphate dehydrogenase. Where indicated, the vertical lines show repositioned gel lanes. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Placenta Growth Factor (PlGF), a Novel Inducer of Plasminogen Activator Inhibitor-1 (PAI-1) in Sickle Cell Disease (SCD) *

    doi: 10.1074/jbc.M110.101691

    Figure Lengend Snippet: PAI-1 expression in mice and human subjects. A and B , plasma levels of PAI-1 in mice as measured by ELISA. Berk-hemi , hemizygous Berkeley mice; Berk-SS , Berkeley SS mice. C , plasma PlGF levels in mice by ELISA. The respective strain is indicated at the bottom of the figure. D , immunohistochemistry for PAI-1 expression in C57BL/6 (normal) and Berkeley SS mouse lungs (shown in brown ; nuclei are stained red ). The top panel shows: (i) antibody control and PAI-1 expression in bronchial epithelial cells of normal and (ii) Berkeley SS mice; the bottom panel shows corresponding staining for PAI-1 in their lung parenchyma. Arrows indicate alveolar macrophages, and arrowheads indicate bronchial epithelial layer. E , the levels of PAI-1 were quantified in BAL of indicated strains of mice by ELISA. F , qRT-PCR analysis of PAI-1 mRNA in MNC from SCD subjects ( n = 9) and normal controls ( n = 9). RQ , relative quantification. G and H , HPMVEC were treated with human recombinant PlGF (250 ng/ml) for the indicated time periods. G , total RNA was subjected to RPA for the expression of indicated genes. H , the culture supernatants were assayed for PAI-1 by ELISA. RPA data are representative of three independent experiments. GAPDH , glyceraldehyde-3-phosphate dehydrogenase. Where indicated, the vertical lines show repositioned gel lanes. *, p

    Article Snippet: The MADPK-71K PAI-1 single plex kit (Millipore Corp., Billerica, MA) was used to assay mouse PAI-1 and read on a Bioplex analyzer (Bio-Rad).

    Techniques: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Quantitative RT-PCR, Recombinant, Recombinase Polymerase Amplification

    Role of PlGF in PAI-1 expression in sickle mice. Berkeley SS mice ( Berk-SS ) were bred with PlGF −/− mice to obtain Berkeley SS/PlGF −/− mice. The plasma levels of PAI-1 are measured by ELISA in the indicated strains of mice ( n = 6).

    Journal: The Journal of Biological Chemistry

    Article Title: Placenta Growth Factor (PlGF), a Novel Inducer of Plasminogen Activator Inhibitor-1 (PAI-1) in Sickle Cell Disease (SCD) *

    doi: 10.1074/jbc.M110.101691

    Figure Lengend Snippet: Role of PlGF in PAI-1 expression in sickle mice. Berkeley SS mice ( Berk-SS ) were bred with PlGF −/− mice to obtain Berkeley SS/PlGF −/− mice. The plasma levels of PAI-1 are measured by ELISA in the indicated strains of mice ( n = 6).

    Article Snippet: The MADPK-71K PAI-1 single plex kit (Millipore Corp., Billerica, MA) was used to assay mouse PAI-1 and read on a Bioplex analyzer (Bio-Rad).

    Techniques: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Identification of functional cis-acting elements in PlGF-induced PAI-1 promoter activation. A , schematics of PAI-1 promoter (−806/+19 bp, relative to transcription start site), indicating the location of HRE-1 to -5, proximal and distal AP-1, C/EBP, and Egr-1 binding sites. Luc , luciferase. B , HPMVEC were co-transfected with either WT or individual HRE mutant ( mut ) constructs of PAI-1 promoter and β-galactosidase plasmid prior to PlGF treatment for 6 h. C , illustrations of an overlapping region (−158/−147 bp) containing the HRE-1 and AP-1 binding sites in the PAI-1 promoter. The mutated oligonucleotides corresponding to HRE-1, AP-1, and the overlapping region of HRE-1 and AP-1 are underlined. D , HPMVEC were co-transfected with either WT or HRE-1 mutant or AP-1 mutant or HRE-1/AP-1 mutant of PAI-1 promoter and β-galactosidase plasmid prior to PlGF treatment for 6 h. The luciferase and β-galactosidase activities were estimated. E , nuclear extracts from untreated and PlGF-treated HPMVEC (10 μg) were incubated either with a biotinylated double-stranded oligonucleotide probe corresponding to an overlapping region containing HRE-1 and AP-1 sites or with a probe containing mutations in an overlapping four shared nucleotides or mutation only in the AP-1 site. Data are representative of three independent experiments. ns , not significant.

    Journal: The Journal of Biological Chemistry

    Article Title: Placenta Growth Factor (PlGF), a Novel Inducer of Plasminogen Activator Inhibitor-1 (PAI-1) in Sickle Cell Disease (SCD) *

    doi: 10.1074/jbc.M110.101691

    Figure Lengend Snippet: Identification of functional cis-acting elements in PlGF-induced PAI-1 promoter activation. A , schematics of PAI-1 promoter (−806/+19 bp, relative to transcription start site), indicating the location of HRE-1 to -5, proximal and distal AP-1, C/EBP, and Egr-1 binding sites. Luc , luciferase. B , HPMVEC were co-transfected with either WT or individual HRE mutant ( mut ) constructs of PAI-1 promoter and β-galactosidase plasmid prior to PlGF treatment for 6 h. C , illustrations of an overlapping region (−158/−147 bp) containing the HRE-1 and AP-1 binding sites in the PAI-1 promoter. The mutated oligonucleotides corresponding to HRE-1, AP-1, and the overlapping region of HRE-1 and AP-1 are underlined. D , HPMVEC were co-transfected with either WT or HRE-1 mutant or AP-1 mutant or HRE-1/AP-1 mutant of PAI-1 promoter and β-galactosidase plasmid prior to PlGF treatment for 6 h. The luciferase and β-galactosidase activities were estimated. E , nuclear extracts from untreated and PlGF-treated HPMVEC (10 μg) were incubated either with a biotinylated double-stranded oligonucleotide probe corresponding to an overlapping region containing HRE-1 and AP-1 sites or with a probe containing mutations in an overlapping four shared nucleotides or mutation only in the AP-1 site. Data are representative of three independent experiments. ns , not significant.

    Article Snippet: The MADPK-71K PAI-1 single plex kit (Millipore Corp., Billerica, MA) was used to assay mouse PAI-1 and read on a Bioplex analyzer (Bio-Rad).

    Techniques: Functional Assay, Activation Assay, Binding Assay, Luciferase, Transfection, Mutagenesis, Construct, Plasmid Preparation, Incubation