fibronectin coated  (Roche)


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    Roche fibronectin coated
    PAK4 Knockdown and Rescue Supports a Kinase-Dependent Role for PAK4 in Podosomes through the Activation of Akt (A) PAK4 shRNAs or scrambled control cells were probed for PAK4 expression. (B) PAK4 shRNA-expressing cells were seeded on <t>fibronectin</t> with TGF-β for 16 h, fixed, and stained for vinculin (green) and F-actin (red). (C) Percentage of cells with podosomes ( > 300 cells per cell line). (D) PAK4 knockdown cells (A) expressing EGFP-tagged shRNA-resistant PAK4: EGFP-PAK4 (rescue) or the kinase dead mutant PAK4: EGFP-PAK4r (K350,351M) were probed for PAK4 expression. (E) Confocal images of PAK4 rescue THP-1 cells. (F) Percentage of cells with podosomes. (G) Cells were probed for pAkt and PAK4. (H) THP-1 cells on fibronectin with TGF-β for 16 h were treated with indicated concentrations of Akt inhibitor for 4 h, fixed and stained for F-actin, and scored for podosomes. (I) Confocal images of THP-1 cells fixed and stained for pAkt, F-actin, and vinculin. Error bars = ± SEM and p values indicate significant differences between treated cells by one-way ANOVA. Scale bars in (B) and (E) = 10 μm; scale bars in (I) = 5 μm.
    Fibronectin Coated, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "PAK4 Kinase Activity Plays a Crucial Role in the Podosome Ring of Myeloid Cells"

    Article Title: PAK4 Kinase Activity Plays a Crucial Role in the Podosome Ring of Myeloid Cells

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2019.11.016

    PAK4 Knockdown and Rescue Supports a Kinase-Dependent Role for PAK4 in Podosomes through the Activation of Akt (A) PAK4 shRNAs or scrambled control cells were probed for PAK4 expression. (B) PAK4 shRNA-expressing cells were seeded on fibronectin with TGF-β for 16 h, fixed, and stained for vinculin (green) and F-actin (red). (C) Percentage of cells with podosomes ( > 300 cells per cell line). (D) PAK4 knockdown cells (A) expressing EGFP-tagged shRNA-resistant PAK4: EGFP-PAK4 (rescue) or the kinase dead mutant PAK4: EGFP-PAK4r (K350,351M) were probed for PAK4 expression. (E) Confocal images of PAK4 rescue THP-1 cells. (F) Percentage of cells with podosomes. (G) Cells were probed for pAkt and PAK4. (H) THP-1 cells on fibronectin with TGF-β for 16 h were treated with indicated concentrations of Akt inhibitor for 4 h, fixed and stained for F-actin, and scored for podosomes. (I) Confocal images of THP-1 cells fixed and stained for pAkt, F-actin, and vinculin. Error bars = ± SEM and p values indicate significant differences between treated cells by one-way ANOVA. Scale bars in (B) and (E) = 10 μm; scale bars in (I) = 5 μm.
    Figure Legend Snippet: PAK4 Knockdown and Rescue Supports a Kinase-Dependent Role for PAK4 in Podosomes through the Activation of Akt (A) PAK4 shRNAs or scrambled control cells were probed for PAK4 expression. (B) PAK4 shRNA-expressing cells were seeded on fibronectin with TGF-β for 16 h, fixed, and stained for vinculin (green) and F-actin (red). (C) Percentage of cells with podosomes ( > 300 cells per cell line). (D) PAK4 knockdown cells (A) expressing EGFP-tagged shRNA-resistant PAK4: EGFP-PAK4 (rescue) or the kinase dead mutant PAK4: EGFP-PAK4r (K350,351M) were probed for PAK4 expression. (E) Confocal images of PAK4 rescue THP-1 cells. (F) Percentage of cells with podosomes. (G) Cells were probed for pAkt and PAK4. (H) THP-1 cells on fibronectin with TGF-β for 16 h were treated with indicated concentrations of Akt inhibitor for 4 h, fixed and stained for F-actin, and scored for podosomes. (I) Confocal images of THP-1 cells fixed and stained for pAkt, F-actin, and vinculin. Error bars = ± SEM and p values indicate significant differences between treated cells by one-way ANOVA. Scale bars in (B) and (E) = 10 μm; scale bars in (I) = 5 μm.

    Techniques Used: Activation Assay, Expressing, shRNA, Staining, Mutagenesis

    PAK4 Localizes to the Podosome Ring (A and B) THP-1 cells stably expressing EGFP or EGFP-PAK4 were seeded on fibronectin with TGF-β for 16 h prior to immunoprecipitation (IP) of paxillin (A) or vinculin (B). Blots were probed for endogenous PAK4 and reprobed for GFP and paxillin or vinculin. (C) EGFP-PAK4-expressing THP-1 cells were seeded on fibronectin with TGF-β for 16 h, fixed, and stained for vinculin. Datasets of 200 images were taken for both EGFP-PAK4 and vinculin and analyzed using the ImageJ 3B plugin. Left panels are reconstructed localizations from two adjacent podosomes (reconstruction blur FWHM = 20 nm), and these localizations are representative of > 50 podosomes analyzed using 3B; scale bar represents 500 nm. Right panel shows a reconstructed dataset from 3B analysis carried out using a computer cluster to give localizations in podosomes of an entire THP-1 cell; scale bar represents 2 μm. (D) STORM and 3B localization of F-actin and EGFP-PAK4, respectively. Scale bar represents 5 μm. (E–G) 3B datasets generated for > 50 podosomes from > 10 EGFP-PAK4-expressing THP-1 cells stained for vinculin (E), paxillin (F), or co-expressing mCherry-Talin (G) were analyzed using ring analysis software ( Staszowska et al., 2017 ). Histograms show the absolute distances from the podosome center of EGFP-PAK4 (blue) and vinculin/paxillin/mCherry-Talin (red). (H) A top-down representation of the relative localizations of PAK4 and the podosome ring proteins vinculin, paxillin, and talin, based on 3B localizations.
    Figure Legend Snippet: PAK4 Localizes to the Podosome Ring (A and B) THP-1 cells stably expressing EGFP or EGFP-PAK4 were seeded on fibronectin with TGF-β for 16 h prior to immunoprecipitation (IP) of paxillin (A) or vinculin (B). Blots were probed for endogenous PAK4 and reprobed for GFP and paxillin or vinculin. (C) EGFP-PAK4-expressing THP-1 cells were seeded on fibronectin with TGF-β for 16 h, fixed, and stained for vinculin. Datasets of 200 images were taken for both EGFP-PAK4 and vinculin and analyzed using the ImageJ 3B plugin. Left panels are reconstructed localizations from two adjacent podosomes (reconstruction blur FWHM = 20 nm), and these localizations are representative of > 50 podosomes analyzed using 3B; scale bar represents 500 nm. Right panel shows a reconstructed dataset from 3B analysis carried out using a computer cluster to give localizations in podosomes of an entire THP-1 cell; scale bar represents 2 μm. (D) STORM and 3B localization of F-actin and EGFP-PAK4, respectively. Scale bar represents 5 μm. (E–G) 3B datasets generated for > 50 podosomes from > 10 EGFP-PAK4-expressing THP-1 cells stained for vinculin (E), paxillin (F), or co-expressing mCherry-Talin (G) were analyzed using ring analysis software ( Staszowska et al., 2017 ). Histograms show the absolute distances from the podosome center of EGFP-PAK4 (blue) and vinculin/paxillin/mCherry-Talin (red). (H) A top-down representation of the relative localizations of PAK4 and the podosome ring proteins vinculin, paxillin, and talin, based on 3B localizations.

    Techniques Used: Stable Transfection, Expressing, Immunoprecipitation, Staining, Generated, Software

    PAK4 Kinase Activity Drives Podosome Formation (A) Confocal images of THP-1 cells seeded on fibronectin with TGF-β for 16 h and then treated for 4 h with DMSO control or 1 μM PAK inhibitors PAK4i or IPA-3. Stained for vinculin (green) and F-actin (red). Insert zoom of podosomes or peripheral adhesions. (B and C) Percentage of THP-1 cells with podosomes following treatment with DMSO or 1 or 5 μM PAK inhibitors for 4 h (B) and the percentage of cells with 0, 1–10, 11–20, 21–30, or ≥31 podosomes per cell was calculated from 300 cells per condition (C). (D) Following a 4 h treatment with DMSO or inhibitors, the inhibitors were washed out and cells incubated with TGF-β for a further 4 h. Cells were fixed and stained for vinculin and F-actin at times indicated, and the percentage of cells with podosomes were counted. (E) Primary human monocytes isolated from peripheral human blood from healthy donors were seeded on fibronectin and differentiated toward macrophages by incubating for 4.5 days with 50 ng/ml M-CSF. Macrophages were then treated with DMSO or 1 μM PAK inhibitor PAK4i or IPA-3, fixed, and stained for vinculin (green) and F-actin (red). (F) Percentage of primary human macrophages with podosomes following a 4 h treatment with DMSO or 1 or 5μM PAK inhibitors. (G) Western blot for PAK1 and PAK4 levels in lysates of primary monocytes from two healthy donors (HD1 and HD2) cultured for 6 days, alongside a THP-1 lysate from cells differentiated for 16 h with TGF-β. For all graphs, error bars represent ±SEM, and p values indicate significant difference to DMSO-treated cells by one-way ANOVA. Scale bars in (A) and (E) represent 10 μm.
    Figure Legend Snippet: PAK4 Kinase Activity Drives Podosome Formation (A) Confocal images of THP-1 cells seeded on fibronectin with TGF-β for 16 h and then treated for 4 h with DMSO control or 1 μM PAK inhibitors PAK4i or IPA-3. Stained for vinculin (green) and F-actin (red). Insert zoom of podosomes or peripheral adhesions. (B and C) Percentage of THP-1 cells with podosomes following treatment with DMSO or 1 or 5 μM PAK inhibitors for 4 h (B) and the percentage of cells with 0, 1–10, 11–20, 21–30, or ≥31 podosomes per cell was calculated from 300 cells per condition (C). (D) Following a 4 h treatment with DMSO or inhibitors, the inhibitors were washed out and cells incubated with TGF-β for a further 4 h. Cells were fixed and stained for vinculin and F-actin at times indicated, and the percentage of cells with podosomes were counted. (E) Primary human monocytes isolated from peripheral human blood from healthy donors were seeded on fibronectin and differentiated toward macrophages by incubating for 4.5 days with 50 ng/ml M-CSF. Macrophages were then treated with DMSO or 1 μM PAK inhibitor PAK4i or IPA-3, fixed, and stained for vinculin (green) and F-actin (red). (F) Percentage of primary human macrophages with podosomes following a 4 h treatment with DMSO or 1 or 5μM PAK inhibitors. (G) Western blot for PAK1 and PAK4 levels in lysates of primary monocytes from two healthy donors (HD1 and HD2) cultured for 6 days, alongside a THP-1 lysate from cells differentiated for 16 h with TGF-β. For all graphs, error bars represent ±SEM, and p values indicate significant difference to DMSO-treated cells by one-way ANOVA. Scale bars in (A) and (E) represent 10 μm.

    Techniques Used: Activity Assay, Indirect Immunoperoxidase Assay, Staining, Incubation, Isolation, Western Blot, Cell Culture

    Podosome Loss Following PAK Inhibition Is Accompanied by an Increase in Focal Adhesions and a Reduction of Invasive Migration (A) For the matrix degradation assay, > 20 fields of view per treatment condition were measured. (B) Mean cell speed (μm/minute) was calculated from > 90 cells from three separate experiments. (C) Confocal images of THP-1 cells seeded on fibronectin with TGF-β for 16 h and then treated for 4 h with 1 μM PAK inhibitors. Cells were stained for zyxin (green), vinculin (red), and F-actin (blue). Insert zoom of peripheral adhesion. Scale bars represent 10 μm. (D) Percentage of cells with focal adhesions. (E) Number of focal adhesions/cell. For all graphs, error bars represent ±SEM, and p values denote significant difference to DMSO-treated cells by one-way ANOVA.
    Figure Legend Snippet: Podosome Loss Following PAK Inhibition Is Accompanied by an Increase in Focal Adhesions and a Reduction of Invasive Migration (A) For the matrix degradation assay, > 20 fields of view per treatment condition were measured. (B) Mean cell speed (μm/minute) was calculated from > 90 cells from three separate experiments. (C) Confocal images of THP-1 cells seeded on fibronectin with TGF-β for 16 h and then treated for 4 h with 1 μM PAK inhibitors. Cells were stained for zyxin (green), vinculin (red), and F-actin (blue). Insert zoom of peripheral adhesion. Scale bars represent 10 μm. (D) Percentage of cells with focal adhesions. (E) Number of focal adhesions/cell. For all graphs, error bars represent ±SEM, and p values denote significant difference to DMSO-treated cells by one-way ANOVA.

    Techniques Used: Inhibition, Migration, Degradation Assay, Staining

    Related Articles

    Lysis:

    Article Title: A deregulated stress response underlies distinct INF2 associated disease profiles
    Article Snippet: .. HEK293T cells were seeded on fibronectin-coated (5 μg/ml in PBS) 10-cm dishes, transfected with INF2 constructs, and harvested by scraping into 1 ml PBS after 24-48 h. After two washing steps with PBS at 500xg for 3 min, cells were resuspended in 200 μl lysis buffer (10 mM Tris/Cl pH 7.5; 150 mM NaCl; 500 µM Ca2+, protease inhibitors (Roche), 2% NP-40). .. In the meantime, 25 μl of GFP-Trap® beads were cleared by washing three times in lysis buffer without NP-40.

    Article Title: PAK4 Kinase Activity Plays a Crucial Role in the Podosome Ring of Myeloid Cells
    Article Snippet: .. Western Blot and Immunoprecipitation Adherent THP-1 cell lysates were made by seeding THP-1 cells on fibronectin-coated 10cm plates with 2ng/ml TGF-β for 16 hours, before washing twice with ice cold PBS and adding 1ml lysis buffer (4M sodium chloride, 1M Tris-HCl pH 7.4, 100mM sodium orthovanadate, 500mM sodium fluoride, 1mM EGTA, 0.5% NP-40, Roche proteinase inhibitor cocktail containing EDTA), then incubating on ice for 10 minutes. .. Cells were then scraped into 1.5ml Eppendorf tubes, before incubating on ice for a further 10 minutes.

    Transfection:

    Article Title: A deregulated stress response underlies distinct INF2 associated disease profiles
    Article Snippet: .. HEK293T cells were seeded on fibronectin-coated (5 μg/ml in PBS) 10-cm dishes, transfected with INF2 constructs, and harvested by scraping into 1 ml PBS after 24-48 h. After two washing steps with PBS at 500xg for 3 min, cells were resuspended in 200 μl lysis buffer (10 mM Tris/Cl pH 7.5; 150 mM NaCl; 500 µM Ca2+, protease inhibitors (Roche), 2% NP-40). .. In the meantime, 25 μl of GFP-Trap® beads were cleared by washing three times in lysis buffer without NP-40.

    Western Blot:

    Article Title: PAK4 Kinase Activity Plays a Crucial Role in the Podosome Ring of Myeloid Cells
    Article Snippet: .. Western Blot and Immunoprecipitation Adherent THP-1 cell lysates were made by seeding THP-1 cells on fibronectin-coated 10cm plates with 2ng/ml TGF-β for 16 hours, before washing twice with ice cold PBS and adding 1ml lysis buffer (4M sodium chloride, 1M Tris-HCl pH 7.4, 100mM sodium orthovanadate, 500mM sodium fluoride, 1mM EGTA, 0.5% NP-40, Roche proteinase inhibitor cocktail containing EDTA), then incubating on ice for 10 minutes. .. Cells were then scraped into 1.5ml Eppendorf tubes, before incubating on ice for a further 10 minutes.

    Construct:

    Article Title: A deregulated stress response underlies distinct INF2 associated disease profiles
    Article Snippet: .. HEK293T cells were seeded on fibronectin-coated (5 μg/ml in PBS) 10-cm dishes, transfected with INF2 constructs, and harvested by scraping into 1 ml PBS after 24-48 h. After two washing steps with PBS at 500xg for 3 min, cells were resuspended in 200 μl lysis buffer (10 mM Tris/Cl pH 7.5; 150 mM NaCl; 500 µM Ca2+, protease inhibitors (Roche), 2% NP-40). .. In the meantime, 25 μl of GFP-Trap® beads were cleared by washing three times in lysis buffer without NP-40.

    Immunoprecipitation:

    Article Title: PAK4 Kinase Activity Plays a Crucial Role in the Podosome Ring of Myeloid Cells
    Article Snippet: .. Western Blot and Immunoprecipitation Adherent THP-1 cell lysates were made by seeding THP-1 cells on fibronectin-coated 10cm plates with 2ng/ml TGF-β for 16 hours, before washing twice with ice cold PBS and adding 1ml lysis buffer (4M sodium chloride, 1M Tris-HCl pH 7.4, 100mM sodium orthovanadate, 500mM sodium fluoride, 1mM EGTA, 0.5% NP-40, Roche proteinase inhibitor cocktail containing EDTA), then incubating on ice for 10 minutes. .. Cells were then scraped into 1.5ml Eppendorf tubes, before incubating on ice for a further 10 minutes.

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    Roche fibronectin coated
    CalDAG GEFI –/– Jurkat T cells show slightly reduced adhesion but unaffected migration properties. Two CalDAG GEFI –/– Jurkat T cell clones were generated and tested for adhesion to ICAM-1 and <t>fibronectin</t> as well as for migration on fibronectin towards a chemoattractant. To rescue adhesion and/or migration abnormalities caused by CalDAG GEFI knock-out, both CalDAG GEFI –/– clones were reconstituted with wildtype CalDAG GEFI. Transduction with empty vector backbone served as treatment control. CalDAG GEFI-competent JE6 Jurkat T cells (JE6, white bars) or CalDAG GEFI –/– clones (no. 1, grey bars, and no. 2, black bars), untransduced (–), empty vector transduced (EV) or CalDAG GEFI reexpressing (WT), were either left unstimulated (unstim) or were stimulated via anti-CD3 (TCR) or MnCl 2 (MnCl 2 ) to induce adhesion to (a) ICAM-1 or (b) fibronectin. (c) JE6 and CalDAG GEFI –/– clones, untransduced and transduced, were tested for migration without chemoattractant (medium) or towards SDF-1α. Data are pooled from three to five independently performed experiments (mean ± SD)
    Fibronectin Coated, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    fibronectin coated - by Bioz Stars, 2021-01
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    CalDAG GEFI –/– Jurkat T cells show slightly reduced adhesion but unaffected migration properties. Two CalDAG GEFI –/– Jurkat T cell clones were generated and tested for adhesion to ICAM-1 and fibronectin as well as for migration on fibronectin towards a chemoattractant. To rescue adhesion and/or migration abnormalities caused by CalDAG GEFI knock-out, both CalDAG GEFI –/– clones were reconstituted with wildtype CalDAG GEFI. Transduction with empty vector backbone served as treatment control. CalDAG GEFI-competent JE6 Jurkat T cells (JE6, white bars) or CalDAG GEFI –/– clones (no. 1, grey bars, and no. 2, black bars), untransduced (–), empty vector transduced (EV) or CalDAG GEFI reexpressing (WT), were either left unstimulated (unstim) or were stimulated via anti-CD3 (TCR) or MnCl 2 (MnCl 2 ) to induce adhesion to (a) ICAM-1 or (b) fibronectin. (c) JE6 and CalDAG GEFI –/– clones, untransduced and transduced, were tested for migration without chemoattractant (medium) or towards SDF-1α. Data are pooled from three to five independently performed experiments (mean ± SD)

    Journal: European Journal of Microbiology & Immunology

    Article Title: The Guanine-Nucleotide Exchange Factor Caldag Gefi Fine-Tunes Functional Properties of Regulatory T Cells

    doi: 10.1556/1886.2017.00007

    Figure Lengend Snippet: CalDAG GEFI –/– Jurkat T cells show slightly reduced adhesion but unaffected migration properties. Two CalDAG GEFI –/– Jurkat T cell clones were generated and tested for adhesion to ICAM-1 and fibronectin as well as for migration on fibronectin towards a chemoattractant. To rescue adhesion and/or migration abnormalities caused by CalDAG GEFI knock-out, both CalDAG GEFI –/– clones were reconstituted with wildtype CalDAG GEFI. Transduction with empty vector backbone served as treatment control. CalDAG GEFI-competent JE6 Jurkat T cells (JE6, white bars) or CalDAG GEFI –/– clones (no. 1, grey bars, and no. 2, black bars), untransduced (–), empty vector transduced (EV) or CalDAG GEFI reexpressing (WT), were either left unstimulated (unstim) or were stimulated via anti-CD3 (TCR) or MnCl 2 (MnCl 2 ) to induce adhesion to (a) ICAM-1 or (b) fibronectin. (c) JE6 and CalDAG GEFI –/– clones, untransduced and transduced, were tested for migration without chemoattractant (medium) or towards SDF-1α. Data are pooled from three to five independently performed experiments (mean ± SD)

    Article Snippet: To study cell adhesion, on the experimental day, cells were stimulated with 5 µg/ml anti-CD3 (OKT3, BD Biosciences), 50 ng/ml PMA (Calbiochem), or 1 mM MnCl2 for 30 min at 37 °C before adhesion on Fc-ICAM-1-coated (0.5 µg/well, R & D system) or Fibronectin-coated (1 µg/well, Roche) 96-well plates.

    Techniques: Migration, Clone Assay, Generated, Knock-Out, Transduction, Plasmid Preparation