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polyclonal goat anti human pdgf dd antibody  (R&D Systems)


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    R&D Systems polyclonal goat anti human pdgf dd antibody
    Figure 1 The basic stretch of amino acids in the conserved putative cleavage site is important for <t>PDGF-DD</t> processing and generation of a PDGFRb agonist. (a) An alignment of the identified cleavage site region in PDGF-C, -R231KSR, with the homologous stretch of amino acids in PDGF-D implies a potential role for the -R254KSK- site in uPA-mediated proteolysis. Conserved basic amino acids are highlighted. (b) An alanine scan mutagenesis assay performed in the conserved basic stretch of amino acids in PDGF-D. Serum-free conditioned media from cells transfected with constructs encoding the depicted mutant PDGF-D(fl) species were incubated with HMWuPA, TCA-precipitated and analysed for processed PDGF-D (arrowhead) by immunoblotting under reducing conditions. (c) In complement to the above experiment, the generated PDGF-DD species were analysed for their ability to induce PDGFRb tyrosine phosphorylation. Serum-free condi- tioned media from cells transfected with constructs encoding the different mutant PDGF-D(fl) species were incubated with HMWu- PA and thereafter applied on PDGFRb-expressing PAE cells. Following cell lysis and specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were determined by immunoblotting under reducing conditions.
    Polyclonal Goat Anti Human Pdgf Dd Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/polyclonal+goat+anti+human+pdgf+dd+antibody/pm18997817-182-8-14?v=R%26D+Systems
    Average 91 stars, based on 18 article reviews
    polyclonal goat anti human pdgf dd antibody - by Bioz Stars, 2026-07
    91/100 stars

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    1) Product Images from "The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth."

    Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

    Journal: Oncogene

    doi: 10.1038/onc.2008.410

    Figure 1 The basic stretch of amino acids in the conserved putative cleavage site is important for PDGF-DD processing and generation of a PDGFRb agonist. (a) An alignment of the identified cleavage site region in PDGF-C, -R231KSR, with the homologous stretch of amino acids in PDGF-D implies a potential role for the -R254KSK- site in uPA-mediated proteolysis. Conserved basic amino acids are highlighted. (b) An alanine scan mutagenesis assay performed in the conserved basic stretch of amino acids in PDGF-D. Serum-free conditioned media from cells transfected with constructs encoding the depicted mutant PDGF-D(fl) species were incubated with HMWuPA, TCA-precipitated and analysed for processed PDGF-D (arrowhead) by immunoblotting under reducing conditions. (c) In complement to the above experiment, the generated PDGF-DD species were analysed for their ability to induce PDGFRb tyrosine phosphorylation. Serum-free condi- tioned media from cells transfected with constructs encoding the different mutant PDGF-D(fl) species were incubated with HMWu- PA and thereafter applied on PDGFRb-expressing PAE cells. Following cell lysis and specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were determined by immunoblotting under reducing conditions.
    Figure Legend Snippet: Figure 1 The basic stretch of amino acids in the conserved putative cleavage site is important for PDGF-DD processing and generation of a PDGFRb agonist. (a) An alignment of the identified cleavage site region in PDGF-C, -R231KSR, with the homologous stretch of amino acids in PDGF-D implies a potential role for the -R254KSK- site in uPA-mediated proteolysis. Conserved basic amino acids are highlighted. (b) An alanine scan mutagenesis assay performed in the conserved basic stretch of amino acids in PDGF-D. Serum-free conditioned media from cells transfected with constructs encoding the depicted mutant PDGF-D(fl) species were incubated with HMWuPA, TCA-precipitated and analysed for processed PDGF-D (arrowhead) by immunoblotting under reducing conditions. (c) In complement to the above experiment, the generated PDGF-DD species were analysed for their ability to induce PDGFRb tyrosine phosphorylation. Serum-free condi- tioned media from cells transfected with constructs encoding the different mutant PDGF-D(fl) species were incubated with HMWu- PA and thereafter applied on PDGFRb-expressing PAE cells. Following cell lysis and specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were determined by immunoblotting under reducing conditions.

    Techniques Used: Mutagenesis, Transfection, Construct, Incubation, Western Blot, Generated, Phospho-proteomics, Expressing, Lysis, Immunoprecipitation

    Figure 2 PDGF-DD interacts directly with uPA. (a) A direct interaction between PDGF-DD and uPA was demonstrated in a Ni-NTA bead assay, where beads coated with PDGF-DD(fl) were incubated with serum-free conditioned medium from uPA-trans- fected cells. Uncoated beads served as negative control. Eluted proteins were analysed by immunoblotting under reducing condi- tions. (b) Specific domain–domain interactions between PDGF-DD and uPA were identified by an ELISA. Serum-free conditioned media from transfected cells expressing the three depicted PDGF- DD species were applied to an ELISA plate coated with HMWuPA and LMWuPA, respectively. Bound PDGF-DD was assayed with specific polyclonal antibodies and visualized using the alkaline phosphatase yellow liquid substrate system. Data are presented as means of triplicates±s.d. after subtraction of absorbance gener- ated by coating proteins and serum-free medium.
    Figure Legend Snippet: Figure 2 PDGF-DD interacts directly with uPA. (a) A direct interaction between PDGF-DD and uPA was demonstrated in a Ni-NTA bead assay, where beads coated with PDGF-DD(fl) were incubated with serum-free conditioned medium from uPA-trans- fected cells. Uncoated beads served as negative control. Eluted proteins were analysed by immunoblotting under reducing condi- tions. (b) Specific domain–domain interactions between PDGF-DD and uPA were identified by an ELISA. Serum-free conditioned media from transfected cells expressing the three depicted PDGF- DD species were applied to an ELISA plate coated with HMWuPA and LMWuPA, respectively. Bound PDGF-DD was assayed with specific polyclonal antibodies and visualized using the alkaline phosphatase yellow liquid substrate system. Data are presented as means of triplicates±s.d. after subtraction of absorbance gener- ated by coating proteins and serum-free medium.

    Techniques Used: Incubation, Negative Control, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Expressing

    Figure 3 PDGF-DD activation by uPA can be modulated by the presence of uPAR. (a) Immunoblotting under reducing conditions of generated PDGF-D species (arrowheads) after in vitro incubation of purified PDGF-DD(fl) with enzymatically comparable levels of LMWuPA and HMWuPA. (b) The PDGF-DD species generated by uPA-mediated proteolysis were analysed for their ability to induce PDGFRb tyrosine phosphorylation. Serum-free conditioned media from cells transfected with PDGF-D(fl) were incubated with LMWuPA or HMWuPA with or without the addition of soluble uPAR (suPAR) and thereafter applied on PDGFRb-expressing PAE cells. Following specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were analysed by immunoblotting under reducing conditions. (c) The ability of scuPA to process PDGF- DD(fl) into active species was explored by applying serum-free conditioned media from cells transfected with PDGF-D(fl) and/or uPA as well as uPAR on PDGFRb-expressing PAE cells. Following cell lysis and specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were analysed by immunoblotting under reducing conditions. To diminish undesired effects generated by plasmin activity induced by the presence of uPA, a2-antiplasmin was included in the serum-free cell culture media after transfection. Also protein levels of uPAR, uPA and PDGF-D(fl) in cells used as transfection host were monitored by immunoblotting under reduced conditions to control transfection efficacy and endogenous levels of protein of interest. Arrowheads indicate the presence of HMWuPA.
    Figure Legend Snippet: Figure 3 PDGF-DD activation by uPA can be modulated by the presence of uPAR. (a) Immunoblotting under reducing conditions of generated PDGF-D species (arrowheads) after in vitro incubation of purified PDGF-DD(fl) with enzymatically comparable levels of LMWuPA and HMWuPA. (b) The PDGF-DD species generated by uPA-mediated proteolysis were analysed for their ability to induce PDGFRb tyrosine phosphorylation. Serum-free conditioned media from cells transfected with PDGF-D(fl) were incubated with LMWuPA or HMWuPA with or without the addition of soluble uPAR (suPAR) and thereafter applied on PDGFRb-expressing PAE cells. Following specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were analysed by immunoblotting under reducing conditions. (c) The ability of scuPA to process PDGF- DD(fl) into active species was explored by applying serum-free conditioned media from cells transfected with PDGF-D(fl) and/or uPA as well as uPAR on PDGFRb-expressing PAE cells. Following cell lysis and specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were analysed by immunoblotting under reducing conditions. To diminish undesired effects generated by plasmin activity induced by the presence of uPA, a2-antiplasmin was included in the serum-free cell culture media after transfection. Also protein levels of uPAR, uPA and PDGF-D(fl) in cells used as transfection host were monitored by immunoblotting under reduced conditions to control transfection efficacy and endogenous levels of protein of interest. Arrowheads indicate the presence of HMWuPA.

    Techniques Used: Activation Assay, Western Blot, Generated, In Vitro, Incubation, Phospho-proteomics, Transfection, Expressing, Immunoprecipitation, Lysis, Activity Assay, Cell Culture, Control

    Figure 4 PDGFRb is regulated by uPAR and facilitates HMWuPA-induced PAE cell migration. (a) Immunoblotting under reduced conditions of cell lysates from semiconfluent cultures to monitor total PDGFRb protein levels in PAE cells with high uPAR expression in comparison to mock-transfected control cells. Calnexin was used as control to ensure equal protein loading. (b) Phosphotyrosine blotting under reducing conditions against immunoprecipitated PDGFRb, following PAE cell stimulation with purified proteins as illustrated. The phosphorylation capacity of PDGFRb in cells with high and low uPAR expression, was compared with mock- transfected PDGFRb-expressing control cells. Cells with low uPAR expression (specifically assigned low expression) were identified in the initial screen of uPAR-expressing clones (data not shown). PDGF-BB was used to monitor phosphorylation induced by another PDGFRb ligand. (c and d) Migration properties of the stably transfected PAE cell lines were determined in a chemotaxis assay. Serum- starved cells were seeded, treated as indicated and allowed to migrate through a porous membrane for 4 h. The response was quantified by cell counting from eight views/membrane and results from one representative experiment are presented as fold migration (migration index) relative to basal migration levels (untreated cells) for each cell line ±s.d. Statistical calculations were carried out with a two- sided Student’s t-test analysis (*Po0.05; ***Po0.001).
    Figure Legend Snippet: Figure 4 PDGFRb is regulated by uPAR and facilitates HMWuPA-induced PAE cell migration. (a) Immunoblotting under reduced conditions of cell lysates from semiconfluent cultures to monitor total PDGFRb protein levels in PAE cells with high uPAR expression in comparison to mock-transfected control cells. Calnexin was used as control to ensure equal protein loading. (b) Phosphotyrosine blotting under reducing conditions against immunoprecipitated PDGFRb, following PAE cell stimulation with purified proteins as illustrated. The phosphorylation capacity of PDGFRb in cells with high and low uPAR expression, was compared with mock- transfected PDGFRb-expressing control cells. Cells with low uPAR expression (specifically assigned low expression) were identified in the initial screen of uPAR-expressing clones (data not shown). PDGF-BB was used to monitor phosphorylation induced by another PDGFRb ligand. (c and d) Migration properties of the stably transfected PAE cell lines were determined in a chemotaxis assay. Serum- starved cells were seeded, treated as indicated and allowed to migrate through a porous membrane for 4 h. The response was quantified by cell counting from eight views/membrane and results from one representative experiment are presented as fold migration (migration index) relative to basal migration levels (untreated cells) for each cell line ±s.d. Statistical calculations were carried out with a two- sided Student’s t-test analysis (*Po0.05; ***Po0.001).

    Techniques Used: Migration, Western Blot, Expressing, Comparison, Transfection, Control, Immunoprecipitation, Cell Stimulation, Phospho-proteomics, Clone Assay, Stable Transfection, Chemotaxis Assay, Membrane, Cell Counting

    Figure 5 Activated PDGF-DD induces cellular transformation of NIH/3T3 cells more effectively than latent PDGF-DD. (a) RT–PCR analysis of PDGF-DD expression in cells stably transfected with PDGF-D(fl) and PDGF-D(GFD), as compared with mock-trans- fected cells. Primers were designed for amplification of the C-terminal part of PDGF-D and b2-microglobulin served as an endogenous reference gene. (b) A cell growth analysis under serum-free conditions monitoring the differences in proliferation capacity of cells stably transfected with equal amounts of PDGF-D(fl) and PDGF-D(GFD), was carried out. Data are presented as means of triplicates±s.d. The experiment was repeated twice. (c) Microphotographs showing representative views of transfected NIH/3T3 cells grown in soft agar to monitor cell number and morphology of the foci generated from cells expressing PDGF-DD(fl) and PDGF-DD(GFD). Quantifications were made from five high magnification pictures (lower panel) and statistical calculations were carried out with a two-sided t-test analysis (**Po0.01). Data are presented as mean number of foci ±s.d. The experiment was repeated twice. Scale bar 0.5mm. (d) Immunofluor- escent staining of permeabilized, fixated NIH/3T3 cells expressing PDGF-DD(fl) and PDGF-DD(GFD), visualizing the presence of uPA (upper panel) and uPAR (lower panel) in transformed cells.
    Figure Legend Snippet: Figure 5 Activated PDGF-DD induces cellular transformation of NIH/3T3 cells more effectively than latent PDGF-DD. (a) RT–PCR analysis of PDGF-DD expression in cells stably transfected with PDGF-D(fl) and PDGF-D(GFD), as compared with mock-trans- fected cells. Primers were designed for amplification of the C-terminal part of PDGF-D and b2-microglobulin served as an endogenous reference gene. (b) A cell growth analysis under serum-free conditions monitoring the differences in proliferation capacity of cells stably transfected with equal amounts of PDGF-D(fl) and PDGF-D(GFD), was carried out. Data are presented as means of triplicates±s.d. The experiment was repeated twice. (c) Microphotographs showing representative views of transfected NIH/3T3 cells grown in soft agar to monitor cell number and morphology of the foci generated from cells expressing PDGF-DD(fl) and PDGF-DD(GFD). Quantifications were made from five high magnification pictures (lower panel) and statistical calculations were carried out with a two-sided t-test analysis (**Po0.01). Data are presented as mean number of foci ±s.d. The experiment was repeated twice. Scale bar 0.5mm. (d) Immunofluor- escent staining of permeabilized, fixated NIH/3T3 cells expressing PDGF-DD(fl) and PDGF-DD(GFD), visualizing the presence of uPA (upper panel) and uPAR (lower panel) in transformed cells.

    Techniques Used: Transformation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Stable Transfection, Transfection, Generated, Staining

    Figure 6 Latent PDGF-DD induces tumour growth in nude mice more effectively than activated PDGF-DD. (a) NIH/3T3 cells stably expressing PDGF-DD(fl) or PDGF-DD(GFD) were xenografted subcutaneously into the midline dorsum of nude mice. Tumour volume was monitored every second day and the experiment was repeated once. Data are presented as mean tumour volume±s.d (PDGF-DD(fl), N ¼ 10; PDGF-DD(GFD), N ¼ 8) starting from day 10 after the majority of the tumours were palpable. Statistical calculations were carried out with a two-sided t-test analysis (*Po0.05; **Po0.01; ***Po0.001). (b) Sections from PDGF-DD derived tumours were stained for PECAM-1/CD31 and detected vessels were quantified by counting nine representative sections/ tumour type and 10 randomly selected views per section. Sizes were categorized as follows: >30 mm large, 10–30 mm medium-sized and o10 mm small. Statistical calculations were carried out with a two- sided Student’s t-test analysis (*Po0.05). Data are presented as mean vessel size ±s.d. (c) Immunohistochemical analysis of sections from tumours expressing PDGF-DD(fl) and PDGF-DD(GFD), was per- formed to confirm abundant PDGF-DD expression and to investigate the expression of uPA and uPAR in both tumour types. Scale bar 25 mm.
    Figure Legend Snippet: Figure 6 Latent PDGF-DD induces tumour growth in nude mice more effectively than activated PDGF-DD. (a) NIH/3T3 cells stably expressing PDGF-DD(fl) or PDGF-DD(GFD) were xenografted subcutaneously into the midline dorsum of nude mice. Tumour volume was monitored every second day and the experiment was repeated once. Data are presented as mean tumour volume±s.d (PDGF-DD(fl), N ¼ 10; PDGF-DD(GFD), N ¼ 8) starting from day 10 after the majority of the tumours were palpable. Statistical calculations were carried out with a two-sided t-test analysis (*Po0.05; **Po0.01; ***Po0.001). (b) Sections from PDGF-DD derived tumours were stained for PECAM-1/CD31 and detected vessels were quantified by counting nine representative sections/ tumour type and 10 randomly selected views per section. Sizes were categorized as follows: >30 mm large, 10–30 mm medium-sized and o10 mm small. Statistical calculations were carried out with a two- sided Student’s t-test analysis (*Po0.05). Data are presented as mean vessel size ±s.d. (c) Immunohistochemical analysis of sections from tumours expressing PDGF-DD(fl) and PDGF-DD(GFD), was per- formed to confirm abundant PDGF-DD expression and to investigate the expression of uPA and uPAR in both tumour types. Scale bar 25 mm.

    Techniques Used: Stable Transfection, Expressing, Derivative Assay, Staining, Immunohistochemical staining

    Figure 7 The pericellular localization of PDGF-DD is controlled by proteolytic processing. (a) NIH/3T3 cells were transiently transfected with PDGF-D(fl) and PDGF-D(GFD), and the pericellular binding of secreted PDGF-DD species was visualized in non-permeabilized (NP) cells by immunofluorescent staining. Permeabilized (P) transfected cells served to ensure equal transfec- tion efficacy. (b) As a control for the experimental setting, NIH/ 3T3 cells were transiently transfected with the two isoforms of VEGF-B. VEGF-B186 is known to be freely diffusible, whereas VEGF-B167 is considered to be strictly matrix-bound. Permeabi- lized (P) transfected cells served to ensure equal transfection efficacy.
    Figure Legend Snippet: Figure 7 The pericellular localization of PDGF-DD is controlled by proteolytic processing. (a) NIH/3T3 cells were transiently transfected with PDGF-D(fl) and PDGF-D(GFD), and the pericellular binding of secreted PDGF-DD species was visualized in non-permeabilized (NP) cells by immunofluorescent staining. Permeabilized (P) transfected cells served to ensure equal transfec- tion efficacy. (b) As a control for the experimental setting, NIH/ 3T3 cells were transiently transfected with the two isoforms of VEGF-B. VEGF-B186 is known to be freely diffusible, whereas VEGF-B167 is considered to be strictly matrix-bound. Permeabi- lized (P) transfected cells served to ensure equal transfection efficacy.

    Techniques Used: Transfection, Binding Assay, Staining, Control



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    polyclonal goat anti human pdgf dd antibody  (R&D Systems)
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    R&D Systems polyclonal goat anti human pdgf dd antibody
    Figure 1 The basic stretch of amino acids in the conserved putative cleavage site is important for <t>PDGF-DD</t> processing and generation of a PDGFRb agonist. (a) An alignment of the identified cleavage site region in PDGF-C, -R231KSR, with the homologous stretch of amino acids in PDGF-D implies a potential role for the -R254KSK- site in uPA-mediated proteolysis. Conserved basic amino acids are highlighted. (b) An alanine scan mutagenesis assay performed in the conserved basic stretch of amino acids in PDGF-D. Serum-free conditioned media from cells transfected with constructs encoding the depicted mutant PDGF-D(fl) species were incubated with HMWuPA, TCA-precipitated and analysed for processed PDGF-D (arrowhead) by immunoblotting under reducing conditions. (c) In complement to the above experiment, the generated PDGF-DD species were analysed for their ability to induce PDGFRb tyrosine phosphorylation. Serum-free condi- tioned media from cells transfected with constructs encoding the different mutant PDGF-D(fl) species were incubated with HMWu- PA and thereafter applied on PDGFRb-expressing PAE cells. Following cell lysis and specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were determined by immunoblotting under reducing conditions.
    Polyclonal Goat Anti Human Pdgf Dd Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/polyclonal+goat+anti+human+pdgf+dd+antibody/pm18997817-182-8-14?v=R%26D+Systems
    Average 91 stars, based on 1 article reviews
    polyclonal goat anti human pdgf dd antibody - by Bioz Stars, 2026-07
    91/100 stars
    		  Buy from Supplier

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    Figure 1 The basic stretch of amino acids in the conserved putative cleavage site is important for PDGF-DD processing and generation of a PDGFRb agonist. (a) An alignment of the identified cleavage site region in PDGF-C, -R231KSR, with the homologous stretch of amino acids in PDGF-D implies a potential role for the -R254KSK- site in uPA-mediated proteolysis. Conserved basic amino acids are highlighted. (b) An alanine scan mutagenesis assay performed in the conserved basic stretch of amino acids in PDGF-D. Serum-free conditioned media from cells transfected with constructs encoding the depicted mutant PDGF-D(fl) species were incubated with HMWuPA, TCA-precipitated and analysed for processed PDGF-D (arrowhead) by immunoblotting under reducing conditions. (c) In complement to the above experiment, the generated PDGF-DD species were analysed for their ability to induce PDGFRb tyrosine phosphorylation. Serum-free condi- tioned media from cells transfected with constructs encoding the different mutant PDGF-D(fl) species were incubated with HMWu- PA and thereafter applied on PDGFRb-expressing PAE cells. Following cell lysis and specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were determined by immunoblotting under reducing conditions.

    Journal: Oncogene

    Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

    doi: 10.1038/onc.2008.410

    Figure Lengend Snippet: Figure 1 The basic stretch of amino acids in the conserved putative cleavage site is important for PDGF-DD processing and generation of a PDGFRb agonist. (a) An alignment of the identified cleavage site region in PDGF-C, -R231KSR, with the homologous stretch of amino acids in PDGF-D implies a potential role for the -R254KSK- site in uPA-mediated proteolysis. Conserved basic amino acids are highlighted. (b) An alanine scan mutagenesis assay performed in the conserved basic stretch of amino acids in PDGF-D. Serum-free conditioned media from cells transfected with constructs encoding the depicted mutant PDGF-D(fl) species were incubated with HMWuPA, TCA-precipitated and analysed for processed PDGF-D (arrowhead) by immunoblotting under reducing conditions. (c) In complement to the above experiment, the generated PDGF-DD species were analysed for their ability to induce PDGFRb tyrosine phosphorylation. Serum-free condi- tioned media from cells transfected with constructs encoding the different mutant PDGF-D(fl) species were incubated with HMWu- PA and thereafter applied on PDGFRb-expressing PAE cells. Following cell lysis and specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were determined by immunoblotting under reducing conditions.

    Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

    Techniques: Mutagenesis, Transfection, Construct, Incubation, Western Blot, Generated, Phospho-proteomics, Expressing, Lysis, Immunoprecipitation

    Figure 2 PDGF-DD interacts directly with uPA. (a) A direct interaction between PDGF-DD and uPA was demonstrated in a Ni-NTA bead assay, where beads coated with PDGF-DD(fl) were incubated with serum-free conditioned medium from uPA-trans- fected cells. Uncoated beads served as negative control. Eluted proteins were analysed by immunoblotting under reducing condi- tions. (b) Specific domain–domain interactions between PDGF-DD and uPA were identified by an ELISA. Serum-free conditioned media from transfected cells expressing the three depicted PDGF- DD species were applied to an ELISA plate coated with HMWuPA and LMWuPA, respectively. Bound PDGF-DD was assayed with specific polyclonal antibodies and visualized using the alkaline phosphatase yellow liquid substrate system. Data are presented as means of triplicates±s.d. after subtraction of absorbance gener- ated by coating proteins and serum-free medium.

    Journal: Oncogene

    Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

    doi: 10.1038/onc.2008.410

    Figure Lengend Snippet: Figure 2 PDGF-DD interacts directly with uPA. (a) A direct interaction between PDGF-DD and uPA was demonstrated in a Ni-NTA bead assay, where beads coated with PDGF-DD(fl) were incubated with serum-free conditioned medium from uPA-trans- fected cells. Uncoated beads served as negative control. Eluted proteins were analysed by immunoblotting under reducing condi- tions. (b) Specific domain–domain interactions between PDGF-DD and uPA were identified by an ELISA. Serum-free conditioned media from transfected cells expressing the three depicted PDGF- DD species were applied to an ELISA plate coated with HMWuPA and LMWuPA, respectively. Bound PDGF-DD was assayed with specific polyclonal antibodies and visualized using the alkaline phosphatase yellow liquid substrate system. Data are presented as means of triplicates±s.d. after subtraction of absorbance gener- ated by coating proteins and serum-free medium.

    Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

    Techniques: Incubation, Negative Control, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Expressing

    Figure 3 PDGF-DD activation by uPA can be modulated by the presence of uPAR. (a) Immunoblotting under reducing conditions of generated PDGF-D species (arrowheads) after in vitro incubation of purified PDGF-DD(fl) with enzymatically comparable levels of LMWuPA and HMWuPA. (b) The PDGF-DD species generated by uPA-mediated proteolysis were analysed for their ability to induce PDGFRb tyrosine phosphorylation. Serum-free conditioned media from cells transfected with PDGF-D(fl) were incubated with LMWuPA or HMWuPA with or without the addition of soluble uPAR (suPAR) and thereafter applied on PDGFRb-expressing PAE cells. Following specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were analysed by immunoblotting under reducing conditions. (c) The ability of scuPA to process PDGF- DD(fl) into active species was explored by applying serum-free conditioned media from cells transfected with PDGF-D(fl) and/or uPA as well as uPAR on PDGFRb-expressing PAE cells. Following cell lysis and specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were analysed by immunoblotting under reducing conditions. To diminish undesired effects generated by plasmin activity induced by the presence of uPA, a2-antiplasmin was included in the serum-free cell culture media after transfection. Also protein levels of uPAR, uPA and PDGF-D(fl) in cells used as transfection host were monitored by immunoblotting under reduced conditions to control transfection efficacy and endogenous levels of protein of interest. Arrowheads indicate the presence of HMWuPA.

    Journal: Oncogene

    Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

    doi: 10.1038/onc.2008.410

    Figure Lengend Snippet: Figure 3 PDGF-DD activation by uPA can be modulated by the presence of uPAR. (a) Immunoblotting under reducing conditions of generated PDGF-D species (arrowheads) after in vitro incubation of purified PDGF-DD(fl) with enzymatically comparable levels of LMWuPA and HMWuPA. (b) The PDGF-DD species generated by uPA-mediated proteolysis were analysed for their ability to induce PDGFRb tyrosine phosphorylation. Serum-free conditioned media from cells transfected with PDGF-D(fl) were incubated with LMWuPA or HMWuPA with or without the addition of soluble uPAR (suPAR) and thereafter applied on PDGFRb-expressing PAE cells. Following specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were analysed by immunoblotting under reducing conditions. (c) The ability of scuPA to process PDGF- DD(fl) into active species was explored by applying serum-free conditioned media from cells transfected with PDGF-D(fl) and/or uPA as well as uPAR on PDGFRb-expressing PAE cells. Following cell lysis and specific immunoprecipitation of PDGFRb, the levels of tyrosine phosphorylation were analysed by immunoblotting under reducing conditions. To diminish undesired effects generated by plasmin activity induced by the presence of uPA, a2-antiplasmin was included in the serum-free cell culture media after transfection. Also protein levels of uPAR, uPA and PDGF-D(fl) in cells used as transfection host were monitored by immunoblotting under reduced conditions to control transfection efficacy and endogenous levels of protein of interest. Arrowheads indicate the presence of HMWuPA.

    Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

    Techniques: Activation Assay, Western Blot, Generated, In Vitro, Incubation, Phospho-proteomics, Transfection, Expressing, Immunoprecipitation, Lysis, Activity Assay, Cell Culture, Control

    Figure 4 PDGFRb is regulated by uPAR and facilitates HMWuPA-induced PAE cell migration. (a) Immunoblotting under reduced conditions of cell lysates from semiconfluent cultures to monitor total PDGFRb protein levels in PAE cells with high uPAR expression in comparison to mock-transfected control cells. Calnexin was used as control to ensure equal protein loading. (b) Phosphotyrosine blotting under reducing conditions against immunoprecipitated PDGFRb, following PAE cell stimulation with purified proteins as illustrated. The phosphorylation capacity of PDGFRb in cells with high and low uPAR expression, was compared with mock- transfected PDGFRb-expressing control cells. Cells with low uPAR expression (specifically assigned low expression) were identified in the initial screen of uPAR-expressing clones (data not shown). PDGF-BB was used to monitor phosphorylation induced by another PDGFRb ligand. (c and d) Migration properties of the stably transfected PAE cell lines were determined in a chemotaxis assay. Serum- starved cells were seeded, treated as indicated and allowed to migrate through a porous membrane for 4 h. The response was quantified by cell counting from eight views/membrane and results from one representative experiment are presented as fold migration (migration index) relative to basal migration levels (untreated cells) for each cell line ±s.d. Statistical calculations were carried out with a two- sided Student’s t-test analysis (*Po0.05; ***Po0.001).

    Journal: Oncogene

    Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

    doi: 10.1038/onc.2008.410

    Figure Lengend Snippet: Figure 4 PDGFRb is regulated by uPAR and facilitates HMWuPA-induced PAE cell migration. (a) Immunoblotting under reduced conditions of cell lysates from semiconfluent cultures to monitor total PDGFRb protein levels in PAE cells with high uPAR expression in comparison to mock-transfected control cells. Calnexin was used as control to ensure equal protein loading. (b) Phosphotyrosine blotting under reducing conditions against immunoprecipitated PDGFRb, following PAE cell stimulation with purified proteins as illustrated. The phosphorylation capacity of PDGFRb in cells with high and low uPAR expression, was compared with mock- transfected PDGFRb-expressing control cells. Cells with low uPAR expression (specifically assigned low expression) were identified in the initial screen of uPAR-expressing clones (data not shown). PDGF-BB was used to monitor phosphorylation induced by another PDGFRb ligand. (c and d) Migration properties of the stably transfected PAE cell lines were determined in a chemotaxis assay. Serum- starved cells were seeded, treated as indicated and allowed to migrate through a porous membrane for 4 h. The response was quantified by cell counting from eight views/membrane and results from one representative experiment are presented as fold migration (migration index) relative to basal migration levels (untreated cells) for each cell line ±s.d. Statistical calculations were carried out with a two- sided Student’s t-test analysis (*Po0.05; ***Po0.001).

    Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

    Techniques: Migration, Western Blot, Expressing, Comparison, Transfection, Control, Immunoprecipitation, Cell Stimulation, Phospho-proteomics, Clone Assay, Stable Transfection, Chemotaxis Assay, Membrane, Cell Counting

    Figure 5 Activated PDGF-DD induces cellular transformation of NIH/3T3 cells more effectively than latent PDGF-DD. (a) RT–PCR analysis of PDGF-DD expression in cells stably transfected with PDGF-D(fl) and PDGF-D(GFD), as compared with mock-trans- fected cells. Primers were designed for amplification of the C-terminal part of PDGF-D and b2-microglobulin served as an endogenous reference gene. (b) A cell growth analysis under serum-free conditions monitoring the differences in proliferation capacity of cells stably transfected with equal amounts of PDGF-D(fl) and PDGF-D(GFD), was carried out. Data are presented as means of triplicates±s.d. The experiment was repeated twice. (c) Microphotographs showing representative views of transfected NIH/3T3 cells grown in soft agar to monitor cell number and morphology of the foci generated from cells expressing PDGF-DD(fl) and PDGF-DD(GFD). Quantifications were made from five high magnification pictures (lower panel) and statistical calculations were carried out with a two-sided t-test analysis (**Po0.01). Data are presented as mean number of foci ±s.d. The experiment was repeated twice. Scale bar 0.5mm. (d) Immunofluor- escent staining of permeabilized, fixated NIH/3T3 cells expressing PDGF-DD(fl) and PDGF-DD(GFD), visualizing the presence of uPA (upper panel) and uPAR (lower panel) in transformed cells.

    Journal: Oncogene

    Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

    doi: 10.1038/onc.2008.410

    Figure Lengend Snippet: Figure 5 Activated PDGF-DD induces cellular transformation of NIH/3T3 cells more effectively than latent PDGF-DD. (a) RT–PCR analysis of PDGF-DD expression in cells stably transfected with PDGF-D(fl) and PDGF-D(GFD), as compared with mock-trans- fected cells. Primers were designed for amplification of the C-terminal part of PDGF-D and b2-microglobulin served as an endogenous reference gene. (b) A cell growth analysis under serum-free conditions monitoring the differences in proliferation capacity of cells stably transfected with equal amounts of PDGF-D(fl) and PDGF-D(GFD), was carried out. Data are presented as means of triplicates±s.d. The experiment was repeated twice. (c) Microphotographs showing representative views of transfected NIH/3T3 cells grown in soft agar to monitor cell number and morphology of the foci generated from cells expressing PDGF-DD(fl) and PDGF-DD(GFD). Quantifications were made from five high magnification pictures (lower panel) and statistical calculations were carried out with a two-sided t-test analysis (**Po0.01). Data are presented as mean number of foci ±s.d. The experiment was repeated twice. Scale bar 0.5mm. (d) Immunofluor- escent staining of permeabilized, fixated NIH/3T3 cells expressing PDGF-DD(fl) and PDGF-DD(GFD), visualizing the presence of uPA (upper panel) and uPAR (lower panel) in transformed cells.

    Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

    Techniques: Transformation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Stable Transfection, Transfection, Generated, Staining

    Figure 6 Latent PDGF-DD induces tumour growth in nude mice more effectively than activated PDGF-DD. (a) NIH/3T3 cells stably expressing PDGF-DD(fl) or PDGF-DD(GFD) were xenografted subcutaneously into the midline dorsum of nude mice. Tumour volume was monitored every second day and the experiment was repeated once. Data are presented as mean tumour volume±s.d (PDGF-DD(fl), N ¼ 10; PDGF-DD(GFD), N ¼ 8) starting from day 10 after the majority of the tumours were palpable. Statistical calculations were carried out with a two-sided t-test analysis (*Po0.05; **Po0.01; ***Po0.001). (b) Sections from PDGF-DD derived tumours were stained for PECAM-1/CD31 and detected vessels were quantified by counting nine representative sections/ tumour type and 10 randomly selected views per section. Sizes were categorized as follows: >30 mm large, 10–30 mm medium-sized and o10 mm small. Statistical calculations were carried out with a two- sided Student’s t-test analysis (*Po0.05). Data are presented as mean vessel size ±s.d. (c) Immunohistochemical analysis of sections from tumours expressing PDGF-DD(fl) and PDGF-DD(GFD), was per- formed to confirm abundant PDGF-DD expression and to investigate the expression of uPA and uPAR in both tumour types. Scale bar 25 mm.

    Journal: Oncogene

    Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

    doi: 10.1038/onc.2008.410

    Figure Lengend Snippet: Figure 6 Latent PDGF-DD induces tumour growth in nude mice more effectively than activated PDGF-DD. (a) NIH/3T3 cells stably expressing PDGF-DD(fl) or PDGF-DD(GFD) were xenografted subcutaneously into the midline dorsum of nude mice. Tumour volume was monitored every second day and the experiment was repeated once. Data are presented as mean tumour volume±s.d (PDGF-DD(fl), N ¼ 10; PDGF-DD(GFD), N ¼ 8) starting from day 10 after the majority of the tumours were palpable. Statistical calculations were carried out with a two-sided t-test analysis (*Po0.05; **Po0.01; ***Po0.001). (b) Sections from PDGF-DD derived tumours were stained for PECAM-1/CD31 and detected vessels were quantified by counting nine representative sections/ tumour type and 10 randomly selected views per section. Sizes were categorized as follows: >30 mm large, 10–30 mm medium-sized and o10 mm small. Statistical calculations were carried out with a two- sided Student’s t-test analysis (*Po0.05). Data are presented as mean vessel size ±s.d. (c) Immunohistochemical analysis of sections from tumours expressing PDGF-DD(fl) and PDGF-DD(GFD), was per- formed to confirm abundant PDGF-DD expression and to investigate the expression of uPA and uPAR in both tumour types. Scale bar 25 mm.

    Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

    Techniques: Stable Transfection, Expressing, Derivative Assay, Staining, Immunohistochemical staining

    Figure 7 The pericellular localization of PDGF-DD is controlled by proteolytic processing. (a) NIH/3T3 cells were transiently transfected with PDGF-D(fl) and PDGF-D(GFD), and the pericellular binding of secreted PDGF-DD species was visualized in non-permeabilized (NP) cells by immunofluorescent staining. Permeabilized (P) transfected cells served to ensure equal transfec- tion efficacy. (b) As a control for the experimental setting, NIH/ 3T3 cells were transiently transfected with the two isoforms of VEGF-B. VEGF-B186 is known to be freely diffusible, whereas VEGF-B167 is considered to be strictly matrix-bound. Permeabi- lized (P) transfected cells served to ensure equal transfection efficacy.

    Journal: Oncogene

    Article Title: The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

    doi: 10.1038/onc.2008.410

    Figure Lengend Snippet: Figure 7 The pericellular localization of PDGF-DD is controlled by proteolytic processing. (a) NIH/3T3 cells were transiently transfected with PDGF-D(fl) and PDGF-D(GFD), and the pericellular binding of secreted PDGF-DD species was visualized in non-permeabilized (NP) cells by immunofluorescent staining. Permeabilized (P) transfected cells served to ensure equal transfec- tion efficacy. (b) As a control for the experimental setting, NIH/ 3T3 cells were transiently transfected with the two isoforms of VEGF-B. VEGF-B186 is known to be freely diffusible, whereas VEGF-B167 is considered to be strictly matrix-bound. Permeabi- lized (P) transfected cells served to ensure equal transfection efficacy.

    Article Snippet: Precipitated proteins were detected by immunoblotting using a polyclonal goat anti-human PDGF-DD antibody (AF1159, RnD Systems).

    Techniques: Transfection, Binding Assay, Staining, Control