affinity chromatography recombinant human hi fgf 2  (GE Healthcare)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    GE Healthcare affinity chromatography recombinant human hi fgf 2
    Human <t>Hi-FGF-2</t> exerts pro-hypertrophic effect. Panel A. Neonatal rat cardiomyocyte cell surface area (normalized, assigning a value of 1 in control, untreated samples) is shown in response to stimulation with Endothelin 1 (ET-1), serving as a positive control, and a recombinant human Hi-FGF-2 preparation (10 ng protein/ml), n = 320 myocytes/group. CM denotes conditioned medium obtained from unstimulated hMFs while CM* denotes conditioned medium from Ang II-stimulated hMFs. ET-1, recombinant human Hi-FGF-2, as well as CM* (but not CM, or Ang II added at 100 nM) increased myocyte cell surface area significantly. Panel B . Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*or CM* supplemented with neutralizing antibodies to total FGF-2 (neu-Ab FGF-2 ), as indicated; n = 480 cells/group. Neutralization of total FGF-2 eliminated the ability of CM* to increase myocytes cell surface area compared to CM. Panel C. Protein synthesis ( 3 H-Leucine incorporation) of cardiomyocytes incubated with CM, CM*, and CM* supplemented with 20 µg/ml neutralizing anti-Hi-FGF-2 antibodies (CM* +neu-Ab Hi-FGF-2 ). Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase protein synthesis of cardiomyocytes compared to CM; n = 5 plates/group. D . Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*, and CM* +neu-Ab Hi-FGF-2 . Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase surface area of cardiomyocytes compared to CM; n = 480/group. Please note that for the experiments shown in B,C,D panels the conditioned media in the first two groups (CM, CM*) were supplemented with non-specific rabbit IgG, at 20 µg/ml. E . Representative images of cardiomyocytes stained for anti-N-cadherin (green), alpha-actinin (red) and nuclei (blue), and incubated with CM, CM*, and CM* +neu-Ab (FGF-2). Sizing bar in (iii) coresponds to 100 µM. In all graphs, brackets show comparison between groups, where *, **, ***, ns correspond to P
    Affinity Chromatography Recombinant Human Hi Fgf 2, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/affinity chromatography recombinant human hi fgf 2/product/GE Healthcare
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    affinity chromatography recombinant human hi fgf 2 - by Bioz Stars, 2020-08
    86/100 stars

    Related Products / Commonly Used Together

    cnbr-activated sepharose
    anti-human hi-fgf-2 antibodies

    Images

    1) Product Images from "High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling"

    Article Title: High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097281

    Human Hi-FGF-2 exerts pro-hypertrophic effect. Panel A. Neonatal rat cardiomyocyte cell surface area (normalized, assigning a value of 1 in control, untreated samples) is shown in response to stimulation with Endothelin 1 (ET-1), serving as a positive control, and a recombinant human Hi-FGF-2 preparation (10 ng protein/ml), n = 320 myocytes/group. CM denotes conditioned medium obtained from unstimulated hMFs while CM* denotes conditioned medium from Ang II-stimulated hMFs. ET-1, recombinant human Hi-FGF-2, as well as CM* (but not CM, or Ang II added at 100 nM) increased myocyte cell surface area significantly. Panel B . Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*or CM* supplemented with neutralizing antibodies to total FGF-2 (neu-Ab FGF-2 ), as indicated; n = 480 cells/group. Neutralization of total FGF-2 eliminated the ability of CM* to increase myocytes cell surface area compared to CM. Panel C. Protein synthesis ( 3 H-Leucine incorporation) of cardiomyocytes incubated with CM, CM*, and CM* supplemented with 20 µg/ml neutralizing anti-Hi-FGF-2 antibodies (CM* +neu-Ab Hi-FGF-2 ). Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase protein synthesis of cardiomyocytes compared to CM; n = 5 plates/group. D . Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*, and CM* +neu-Ab Hi-FGF-2 . Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase surface area of cardiomyocytes compared to CM; n = 480/group. Please note that for the experiments shown in B,C,D panels the conditioned media in the first two groups (CM, CM*) were supplemented with non-specific rabbit IgG, at 20 µg/ml. E . Representative images of cardiomyocytes stained for anti-N-cadherin (green), alpha-actinin (red) and nuclei (blue), and incubated with CM, CM*, and CM* +neu-Ab (FGF-2). Sizing bar in (iii) coresponds to 100 µM. In all graphs, brackets show comparison between groups, where *, **, ***, ns correspond to P
    Figure Legend Snippet: Human Hi-FGF-2 exerts pro-hypertrophic effect. Panel A. Neonatal rat cardiomyocyte cell surface area (normalized, assigning a value of 1 in control, untreated samples) is shown in response to stimulation with Endothelin 1 (ET-1), serving as a positive control, and a recombinant human Hi-FGF-2 preparation (10 ng protein/ml), n = 320 myocytes/group. CM denotes conditioned medium obtained from unstimulated hMFs while CM* denotes conditioned medium from Ang II-stimulated hMFs. ET-1, recombinant human Hi-FGF-2, as well as CM* (but not CM, or Ang II added at 100 nM) increased myocyte cell surface area significantly. Panel B . Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*or CM* supplemented with neutralizing antibodies to total FGF-2 (neu-Ab FGF-2 ), as indicated; n = 480 cells/group. Neutralization of total FGF-2 eliminated the ability of CM* to increase myocytes cell surface area compared to CM. Panel C. Protein synthesis ( 3 H-Leucine incorporation) of cardiomyocytes incubated with CM, CM*, and CM* supplemented with 20 µg/ml neutralizing anti-Hi-FGF-2 antibodies (CM* +neu-Ab Hi-FGF-2 ). Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase protein synthesis of cardiomyocytes compared to CM; n = 5 plates/group. D . Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*, and CM* +neu-Ab Hi-FGF-2 . Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase surface area of cardiomyocytes compared to CM; n = 480/group. Please note that for the experiments shown in B,C,D panels the conditioned media in the first two groups (CM, CM*) were supplemented with non-specific rabbit IgG, at 20 µg/ml. E . Representative images of cardiomyocytes stained for anti-N-cadherin (green), alpha-actinin (red) and nuclei (blue), and incubated with CM, CM*, and CM* +neu-Ab (FGF-2). Sizing bar in (iii) coresponds to 100 µM. In all graphs, brackets show comparison between groups, where *, **, ***, ns correspond to P

    Techniques Used: Positive Control, Recombinant, Incubation, Neutralization, Staining

    Detection of Hi-FGF-2 in human atrial tissue. Panel (A) shows representative western blot images of human atrial extracts (hA1, hA2, hA3, 50 µg/lane) probed for FGF-2 with an antibody detecting all FGF-2 isoforms. Expected migration of all human FGF-2 isoforms (34, 24, 22–22.5, and 18 kDa), corresponding to Hi- or Lo-FGF-2, is indicated by arrows; please note that the 34 kDa isoform is not detectable in tissue lysates. Western blots were also probed for cardiac troponin T (TnT) to verify equivalent loading of lanes. Samples hA1, hA2 were analyzed in small (8.3×5.5 cm 2 ) 15% polyacrylamide gels, while hA3 was analyzed in a large (16×11.5 cm 2 ) 15% polyacrylamide gel. The included graph shows percentage of each isoform over total FGF-2, where, n = 45; comparisons between groups are indicated by brackets, where *** and ** denote P
    Figure Legend Snippet: Detection of Hi-FGF-2 in human atrial tissue. Panel (A) shows representative western blot images of human atrial extracts (hA1, hA2, hA3, 50 µg/lane) probed for FGF-2 with an antibody detecting all FGF-2 isoforms. Expected migration of all human FGF-2 isoforms (34, 24, 22–22.5, and 18 kDa), corresponding to Hi- or Lo-FGF-2, is indicated by arrows; please note that the 34 kDa isoform is not detectable in tissue lysates. Western blots were also probed for cardiac troponin T (TnT) to verify equivalent loading of lanes. Samples hA1, hA2 were analyzed in small (8.3×5.5 cm 2 ) 15% polyacrylamide gels, while hA3 was analyzed in a large (16×11.5 cm 2 ) 15% polyacrylamide gel. The included graph shows percentage of each isoform over total FGF-2, where, n = 45; comparisons between groups are indicated by brackets, where *** and ** denote P

    Techniques Used: Western Blot, Migration

    Detection of Hi-FGF-2 in human atrial myofibroblasts. Panel (A) shows two sets of western blots analyzing FGF-2 isoforms. The first set, (i), is a composite of two blots (separated by a broken line) and analyzes FGF-2 isoforms in hMF lysates (20 µg/lane), from atrial myofibroblast primary cultures obtained from 10 patients (patients 11–20), and correspondingly labeled as C11–20. The second set, (ii), also a composite of two blots separated by a broken line, analyzes FGF-2 isoforms in atrial tissue lysates from patients 11–20, and labelled T11–20 (50 µg/lane). The hMF blots or tissue blots were also probed for, respectively, β-tubulin (β-tub), or Troponin-T (TnT), as indicated. Following densitometry of the hMF blots, the % contribution of each FGF-2 isoform to the total FGF-2 signal was determined for each individual lane, and cumulative results (mean±SD) are included in graph form (n = 10). In Panel (B) , a western blot shows FGF-2 signals from 0.5 and 0.2 ng/lane of recombinant histidine tagged Lo-FGF-2 (FGF-2 His ), atrial tissue lysates (T11, T15 and T17, loaded at 50 µg/lane), side by side with FGF-2 signals from lysates obtained from corresponding primary hMF cultures (C11, C15 and C17, loaded at 10 µg/lane). The graph shows comparisons between tissue and cell lysates for their relative total FGF-2 content, assessed by densitometry as optical density (O.D.) units (n = 3). Measurements corresponding to cell FGF-2 were multiplied by 5, to correct for the 5-fold difference in total protein loading. In both panels, comparisons between groups are indicated by brackets, where P > 0.05 is marked as ns, while P
    Figure Legend Snippet: Detection of Hi-FGF-2 in human atrial myofibroblasts. Panel (A) shows two sets of western blots analyzing FGF-2 isoforms. The first set, (i), is a composite of two blots (separated by a broken line) and analyzes FGF-2 isoforms in hMF lysates (20 µg/lane), from atrial myofibroblast primary cultures obtained from 10 patients (patients 11–20), and correspondingly labeled as C11–20. The second set, (ii), also a composite of two blots separated by a broken line, analyzes FGF-2 isoforms in atrial tissue lysates from patients 11–20, and labelled T11–20 (50 µg/lane). The hMF blots or tissue blots were also probed for, respectively, β-tubulin (β-tub), or Troponin-T (TnT), as indicated. Following densitometry of the hMF blots, the % contribution of each FGF-2 isoform to the total FGF-2 signal was determined for each individual lane, and cumulative results (mean±SD) are included in graph form (n = 10). In Panel (B) , a western blot shows FGF-2 signals from 0.5 and 0.2 ng/lane of recombinant histidine tagged Lo-FGF-2 (FGF-2 His ), atrial tissue lysates (T11, T15 and T17, loaded at 50 µg/lane), side by side with FGF-2 signals from lysates obtained from corresponding primary hMF cultures (C11, C15 and C17, loaded at 10 µg/lane). The graph shows comparisons between tissue and cell lysates for their relative total FGF-2 content, assessed by densitometry as optical density (O.D.) units (n = 3). Measurements corresponding to cell FGF-2 were multiplied by 5, to correct for the 5-fold difference in total protein loading. In both panels, comparisons between groups are indicated by brackets, where P > 0.05 is marked as ns, while P

    Techniques Used: Western Blot, Labeling, Recombinant

    Selective neutralization of extracellular human Hi-FGF-2 attenuates expression of pro-fibrotic proteins. Panel A . Western blots showing the effect of incubation with either control antibodies (Cont-Ab, 20 µg/ml, lanes 1,2,3), or anti-Hi-FGF-2 antibodies (Neu-Ab Hi-FGF-2 , 20 µg/ml, lanes 4,5,6) on the accumulation of α-SMA, procollagen, SMemb, EDA-Fibronectin (EDA-FN), β-tubulin, and GAPDH, by hMFs, as indicated. Panels B , C , D and E show corresponding quantitative (densitometry) data for α-SMA, procollagen, SMemb, EDA-Fibronectin (EDA-FN), as indicated (±SEM). Incubation with Neu-Ab Hi-FGF-2 significantly decreased expression of α-SMA, procollagen, SMemb and EDA-Fibronectin, without having any effect on GAPDH or β-tubulin. Brackets show comparisons between groups, where *, **, *** correspond to P
    Figure Legend Snippet: Selective neutralization of extracellular human Hi-FGF-2 attenuates expression of pro-fibrotic proteins. Panel A . Western blots showing the effect of incubation with either control antibodies (Cont-Ab, 20 µg/ml, lanes 1,2,3), or anti-Hi-FGF-2 antibodies (Neu-Ab Hi-FGF-2 , 20 µg/ml, lanes 4,5,6) on the accumulation of α-SMA, procollagen, SMemb, EDA-Fibronectin (EDA-FN), β-tubulin, and GAPDH, by hMFs, as indicated. Panels B , C , D and E show corresponding quantitative (densitometry) data for α-SMA, procollagen, SMemb, EDA-Fibronectin (EDA-FN), as indicated (±SEM). Incubation with Neu-Ab Hi-FGF-2 significantly decreased expression of α-SMA, procollagen, SMemb and EDA-Fibronectin, without having any effect on GAPDH or β-tubulin. Brackets show comparisons between groups, where *, **, *** correspond to P

    Techniques Used: Neutralization, Expressing, Western Blot, Incubation

    Effect of extracellular-acting FGF-2 isoforms on the accumulation of pro-IL-1β and PAI-1 by hMFs. Panel A , western blot and corresponding cumulative data showing relative pro-IL-1β levels (optical density, O.D. units) in hMF cell lysates from unstimulated cells (lanes 1,2,3) and cells stimulated with 10 ng/ml of a recombinant Hi-FGF-2 preparation (Hi, lanes 4,5,6) or 10 ng/ml of recombinant Lo-FGF-2 (Lo, lanes 7,8,9), as indicated. Both Hi- and Lo-FGF-2 upregulated pro-IL-1β, although the effect of Hi-FGF-2 was significantly more potent. Panel B , western blot and corresponding quantitative data showing relative PAI-1 levels (optical density, O.D. units) in hMF cell lysates from unstimulated cells (lanes 1,2,3) and cells stimulated with Hi-FGF-2 (Hi, lanes 4,5,6) or Lo-FGF-2 (Lo, lanes 7,8,9), as indicated. Hi- but not Lo-FGF-2 upregulated PAI-1 levels. Brackets mark comparisons between groups where *, **, ***, and ns denotes P
    Figure Legend Snippet: Effect of extracellular-acting FGF-2 isoforms on the accumulation of pro-IL-1β and PAI-1 by hMFs. Panel A , western blot and corresponding cumulative data showing relative pro-IL-1β levels (optical density, O.D. units) in hMF cell lysates from unstimulated cells (lanes 1,2,3) and cells stimulated with 10 ng/ml of a recombinant Hi-FGF-2 preparation (Hi, lanes 4,5,6) or 10 ng/ml of recombinant Lo-FGF-2 (Lo, lanes 7,8,9), as indicated. Both Hi- and Lo-FGF-2 upregulated pro-IL-1β, although the effect of Hi-FGF-2 was significantly more potent. Panel B , western blot and corresponding quantitative data showing relative PAI-1 levels (optical density, O.D. units) in hMF cell lysates from unstimulated cells (lanes 1,2,3) and cells stimulated with Hi-FGF-2 (Hi, lanes 4,5,6) or Lo-FGF-2 (Lo, lanes 7,8,9), as indicated. Hi- but not Lo-FGF-2 upregulated PAI-1 levels. Brackets mark comparisons between groups where *, **, ***, and ns denotes P

    Techniques Used: Western Blot, Recombinant

    Detection of Hi-FGF-2 in the extracellular environment in vitro and in vivo . Panel (A) . Representative western blot images of FGF-2 detection in conditioned medium from unstimulated or Ang II-stimulated hMFs. Each lane contains the heparin-sepharose-bound fraction from 60 ml of pooled conditioned medium. Panel (B) . Representative western blots for FGF-2 “eluted” from the cell surface with a high salt wash, and concentrated by binding to heparin-sepharose. Each lane contains the heparin-bound fraction from a 10 ml wash (5×100 near-confluent plates). Ponceau S Red (P-Red) staining of unidentified protein band(s) is also shown, indicative of equivalent loading. Experiments shown in A and B were repeated 2 more times, with similar results. Panel (C) . Western blot image, and corresponding quantitative data of FGF-2 isoforms present in human pericardial fluid (n = 10). Lanes 1-5 (gel 1) and 6-10 (gel 2) contain the heparin-sepharose-bound fraction from 0.5 ml pericardial fluid of individual patients. Lanes marked as F1, F2 contain recombinant Lo-FGF-2 (histidine-tagged) loaded at 0.25 and 0.5 ng/lane respectively. Sample 10 was deliberately overexposed to increase visibility of bands. Recombinant FGF-2, used as standard, was included in the second gel as well (not shown here). The graph shows percent contribution of Hi- or Lo-FGF-2 isoforms to the total FGF-2 signal (mean ± SEM). In all panels, brackets show comparisons between groups; * and ** correspond to P
    Figure Legend Snippet: Detection of Hi-FGF-2 in the extracellular environment in vitro and in vivo . Panel (A) . Representative western blot images of FGF-2 detection in conditioned medium from unstimulated or Ang II-stimulated hMFs. Each lane contains the heparin-sepharose-bound fraction from 60 ml of pooled conditioned medium. Panel (B) . Representative western blots for FGF-2 “eluted” from the cell surface with a high salt wash, and concentrated by binding to heparin-sepharose. Each lane contains the heparin-bound fraction from a 10 ml wash (5×100 near-confluent plates). Ponceau S Red (P-Red) staining of unidentified protein band(s) is also shown, indicative of equivalent loading. Experiments shown in A and B were repeated 2 more times, with similar results. Panel (C) . Western blot image, and corresponding quantitative data of FGF-2 isoforms present in human pericardial fluid (n = 10). Lanes 1-5 (gel 1) and 6-10 (gel 2) contain the heparin-sepharose-bound fraction from 0.5 ml pericardial fluid of individual patients. Lanes marked as F1, F2 contain recombinant Lo-FGF-2 (histidine-tagged) loaded at 0.25 and 0.5 ng/lane respectively. Sample 10 was deliberately overexposed to increase visibility of bands. Recombinant FGF-2, used as standard, was included in the second gel as well (not shown here). The graph shows percent contribution of Hi- or Lo-FGF-2 isoforms to the total FGF-2 signal (mean ± SEM). In all panels, brackets show comparisons between groups; * and ** correspond to P

    Techniques Used: In Vitro, In Vivo, Western Blot, Binding Assay, Staining, Recombinant

    ERK and MMP-2 activities mediate the Ang II-induced Hi-FGF-2 upregulation in hMFs. Panel A. Western blot and corresponding cumulative data showing the effect of an ERK inhibitor (U0126), or MMP-2 inhibitor (MMP2i) on the Ang II induced Hi-FGF-2 upregulation. Signal for β-tubulin is also shown, serving as loading control. Panel B. Western blots and corresponding cumulative data showing the effect of Ang II administration on phospho-(P)-ERK and total ERK, after 10–30 minutes and 6–24 hours of stimulation as indicated. The graph shows cumulative data (n = 3) of the ratio between P-ERK/ERK over time (10–30 min, 6–24 hours), in response to Ang II. Minutes and hours are indicated as ‘ and h. Panel (C) Representative zymogram of MMP-2 activity in hMFs, including a positive control band (MMP-2), and corresponding cumulative data, showing relative MMP-2 activity in response to Ang II, over time (10–30 min, 6–24 hours), as indicated. For all graphs, brackets show comparisons between groups; *, **, ***, and ns correspond to P
    Figure Legend Snippet: ERK and MMP-2 activities mediate the Ang II-induced Hi-FGF-2 upregulation in hMFs. Panel A. Western blot and corresponding cumulative data showing the effect of an ERK inhibitor (U0126), or MMP-2 inhibitor (MMP2i) on the Ang II induced Hi-FGF-2 upregulation. Signal for β-tubulin is also shown, serving as loading control. Panel B. Western blots and corresponding cumulative data showing the effect of Ang II administration on phospho-(P)-ERK and total ERK, after 10–30 minutes and 6–24 hours of stimulation as indicated. The graph shows cumulative data (n = 3) of the ratio between P-ERK/ERK over time (10–30 min, 6–24 hours), in response to Ang II. Minutes and hours are indicated as ‘ and h. Panel (C) Representative zymogram of MMP-2 activity in hMFs, including a positive control band (MMP-2), and corresponding cumulative data, showing relative MMP-2 activity in response to Ang II, over time (10–30 min, 6–24 hours), as indicated. For all graphs, brackets show comparisons between groups; *, **, ***, and ns correspond to P

    Techniques Used: Western Blot, Activity Assay, Positive Control

    Both AT-1R and AT-2R mediate the Ang II-induced ERK activation in hMFs. Panel A shows western blot of activated (phosphorylated) pERK, and total ERK, in hMFs stimulated for 30 minutes with with Ang II (lanes 1,2,3), Ang II + PD123319 (lanes 4,5,6), Ang II + Losartan (lanes 7,8,9), and Ang II +PD123319 +Losartan (lanes 10,11,12), in the absence (-) or presence (+) of neutralizing anti-FGF-2 antibodies (neu-Ab FGF-2 ), as indicated. Please note that the western blot for pERK in the groups incubated with neu-Ab FGF-2 is not directly comparable to the western blot for pERK in the groups incubated in the absence of neu-Ab FGF-2 (different exposures). Panel B shows pERK/ERK ratios in the groups shown in panel A. Brackets show statistically significant differences between groups, where *, **, ***, correspond to P
    Figure Legend Snippet: Both AT-1R and AT-2R mediate the Ang II-induced ERK activation in hMFs. Panel A shows western blot of activated (phosphorylated) pERK, and total ERK, in hMFs stimulated for 30 minutes with with Ang II (lanes 1,2,3), Ang II + PD123319 (lanes 4,5,6), Ang II + Losartan (lanes 7,8,9), and Ang II +PD123319 +Losartan (lanes 10,11,12), in the absence (-) or presence (+) of neutralizing anti-FGF-2 antibodies (neu-Ab FGF-2 ), as indicated. Please note that the western blot for pERK in the groups incubated with neu-Ab FGF-2 is not directly comparable to the western blot for pERK in the groups incubated in the absence of neu-Ab FGF-2 (different exposures). Panel B shows pERK/ERK ratios in the groups shown in panel A. Brackets show statistically significant differences between groups, where *, **, ***, correspond to P

    Techniques Used: Activation Assay, Western Blot, Incubation

    Angiotensin II promotes upregulation of cell-associated human Hi-FGF-2 via AT-1R and AT-2R. Panel A : western blot, and corresponding cumulative data, showing the effect of Ang II on Hi-FGF-2 accumulation by hMFs, in the absence or presence of either losartan (AT-1R inhibitor) or PD123319 (AT-2R inhibitor). Lanes 1–3; 4–6; 7–9; 10–12 correspond to lysates from, respectively, untreated (Control)-;Ang II-stimulated-; Ang II stimulated in the presence of losartan; and Ang II-stimulated in the presence of PD123319- hMFs. Ang II promotes Hi-FGF-2 upregulation which is significantly decreased by either losartan or PD123319. Panel B : western blot and cumulative densitometry data showing the effect of Ang II on Hi-FGF-2 accumulation in the absence or presence of simultaneous inhibition of both AT-1R and AT-2R. Lanes 1–3; 4–6; 7–9 correspond to lysates from, respectively, untreated (Control)-;Ang II-stimulated-; Ang II stimulated in the presence of both losartan and PD123319- hMFs. Relative levels of Hi-FGF-2 in the presence of both AT-1R and AT-2R inhibitors are not significantly different to those of unstimulated cells. Panels C and D . Western blots showing expression, respectively, of AT-1R or AT-2R by hMFs, and relative levels of these receptors after 24 h stimulation with Ang II. After 24 hour stimulation, levels of AT-1R, but not AT-R2, decrease compared to unstimulated cells. Signal for β-actin is also shown in A-D, serving as loading control. E . Densitometry data showing the effect of Ang II receptor inhibitors on baseline Hi-FGF-2 accumulation by hMFs in the abcence of stimulation by added Ang II. Incubation of unstimulated hMFs with losartan (but not PD123319) significantly decreased baseline Hi-FGF-2 levels. Sample size n = 3 (all graphs); *, **, *** indicates P
    Figure Legend Snippet: Angiotensin II promotes upregulation of cell-associated human Hi-FGF-2 via AT-1R and AT-2R. Panel A : western blot, and corresponding cumulative data, showing the effect of Ang II on Hi-FGF-2 accumulation by hMFs, in the absence or presence of either losartan (AT-1R inhibitor) or PD123319 (AT-2R inhibitor). Lanes 1–3; 4–6; 7–9; 10–12 correspond to lysates from, respectively, untreated (Control)-;Ang II-stimulated-; Ang II stimulated in the presence of losartan; and Ang II-stimulated in the presence of PD123319- hMFs. Ang II promotes Hi-FGF-2 upregulation which is significantly decreased by either losartan or PD123319. Panel B : western blot and cumulative densitometry data showing the effect of Ang II on Hi-FGF-2 accumulation in the absence or presence of simultaneous inhibition of both AT-1R and AT-2R. Lanes 1–3; 4–6; 7–9 correspond to lysates from, respectively, untreated (Control)-;Ang II-stimulated-; Ang II stimulated in the presence of both losartan and PD123319- hMFs. Relative levels of Hi-FGF-2 in the presence of both AT-1R and AT-2R inhibitors are not significantly different to those of unstimulated cells. Panels C and D . Western blots showing expression, respectively, of AT-1R or AT-2R by hMFs, and relative levels of these receptors after 24 h stimulation with Ang II. After 24 hour stimulation, levels of AT-1R, but not AT-R2, decrease compared to unstimulated cells. Signal for β-actin is also shown in A-D, serving as loading control. E . Densitometry data showing the effect of Ang II receptor inhibitors on baseline Hi-FGF-2 accumulation by hMFs in the abcence of stimulation by added Ang II. Incubation of unstimulated hMFs with losartan (but not PD123319) significantly decreased baseline Hi-FGF-2 levels. Sample size n = 3 (all graphs); *, **, *** indicates P

    Techniques Used: Western Blot, Inhibition, Expressing, Incubation

    Related Articles

    Isolation:

    Article Title: High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling
    Article Snippet: .. Isolation of anti-human Hi-FGF-2 antibodies by affinity chromatography Recombinant human Hi-FGF-2 (24 kDa isoform) was cross-linked to CNBr-activated Sepharose (GE Healthcare), as per manufacturer's instructions. .. The rabbit anti-Hi-FGF-2 antiserum (1 ml) was diluted with 9 ml binding buffer recombinant (40 mM Tris-HCl pH 8.0, 0.2 M NaCl), clarified by filtration, and incubated with human Hi-FGF-2-Sepharose (1 ml slurry) at ambient temperature, with gentle shaking for 2 hours.

    Affinity Chromatography:

    Article Title: High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling
    Article Snippet: .. Isolation of anti-human Hi-FGF-2 antibodies by affinity chromatography Recombinant human Hi-FGF-2 (24 kDa isoform) was cross-linked to CNBr-activated Sepharose (GE Healthcare), as per manufacturer's instructions. .. The rabbit anti-Hi-FGF-2 antiserum (1 ml) was diluted with 9 ml binding buffer recombinant (40 mM Tris-HCl pH 8.0, 0.2 M NaCl), clarified by filtration, and incubated with human Hi-FGF-2-Sepharose (1 ml slurry) at ambient temperature, with gentle shaking for 2 hours.

    Recombinant:

    Article Title: High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling
    Article Snippet: .. Isolation of anti-human Hi-FGF-2 antibodies by affinity chromatography Recombinant human Hi-FGF-2 (24 kDa isoform) was cross-linked to CNBr-activated Sepharose (GE Healthcare), as per manufacturer's instructions. .. The rabbit anti-Hi-FGF-2 antiserum (1 ml) was diluted with 9 ml binding buffer recombinant (40 mM Tris-HCl pH 8.0, 0.2 M NaCl), clarified by filtration, and incubated with human Hi-FGF-2-Sepharose (1 ml slurry) at ambient temperature, with gentle shaking for 2 hours.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    GE Healthcare affinity chromatography recombinant human hi fgf 2
    Human <t>Hi-FGF-2</t> exerts pro-hypertrophic effect. Panel A. Neonatal rat cardiomyocyte cell surface area (normalized, assigning a value of 1 in control, untreated samples) is shown in response to stimulation with Endothelin 1 (ET-1), serving as a positive control, and a recombinant human Hi-FGF-2 preparation (10 ng protein/ml), n = 320 myocytes/group. CM denotes conditioned medium obtained from unstimulated hMFs while CM* denotes conditioned medium from Ang II-stimulated hMFs. ET-1, recombinant human Hi-FGF-2, as well as CM* (but not CM, or Ang II added at 100 nM) increased myocyte cell surface area significantly. Panel B . Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*or CM* supplemented with neutralizing antibodies to total FGF-2 (neu-Ab FGF-2 ), as indicated; n = 480 cells/group. Neutralization of total FGF-2 eliminated the ability of CM* to increase myocytes cell surface area compared to CM. Panel C. Protein synthesis ( 3 H-Leucine incorporation) of cardiomyocytes incubated with CM, CM*, and CM* supplemented with 20 µg/ml neutralizing anti-Hi-FGF-2 antibodies (CM* +neu-Ab Hi-FGF-2 ). Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase protein synthesis of cardiomyocytes compared to CM; n = 5 plates/group. D . Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*, and CM* +neu-Ab Hi-FGF-2 . Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase surface area of cardiomyocytes compared to CM; n = 480/group. Please note that for the experiments shown in B,C,D panels the conditioned media in the first two groups (CM, CM*) were supplemented with non-specific rabbit IgG, at 20 µg/ml. E . Representative images of cardiomyocytes stained for anti-N-cadherin (green), alpha-actinin (red) and nuclei (blue), and incubated with CM, CM*, and CM* +neu-Ab (FGF-2). Sizing bar in (iii) coresponds to 100 µM. In all graphs, brackets show comparison between groups, where *, **, ***, ns correspond to P
    Affinity Chromatography Recombinant Human Hi Fgf 2, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/affinity chromatography recombinant human hi fgf 2/product/GE Healthcare
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    affinity chromatography recombinant human hi fgf 2 - by Bioz Stars, 2020-08
    86/100 stars
      Buy from Supplier

    Image Search Results


    Human Hi-FGF-2 exerts pro-hypertrophic effect. Panel A. Neonatal rat cardiomyocyte cell surface area (normalized, assigning a value of 1 in control, untreated samples) is shown in response to stimulation with Endothelin 1 (ET-1), serving as a positive control, and a recombinant human Hi-FGF-2 preparation (10 ng protein/ml), n = 320 myocytes/group. CM denotes conditioned medium obtained from unstimulated hMFs while CM* denotes conditioned medium from Ang II-stimulated hMFs. ET-1, recombinant human Hi-FGF-2, as well as CM* (but not CM, or Ang II added at 100 nM) increased myocyte cell surface area significantly. Panel B . Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*or CM* supplemented with neutralizing antibodies to total FGF-2 (neu-Ab FGF-2 ), as indicated; n = 480 cells/group. Neutralization of total FGF-2 eliminated the ability of CM* to increase myocytes cell surface area compared to CM. Panel C. Protein synthesis ( 3 H-Leucine incorporation) of cardiomyocytes incubated with CM, CM*, and CM* supplemented with 20 µg/ml neutralizing anti-Hi-FGF-2 antibodies (CM* +neu-Ab Hi-FGF-2 ). Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase protein synthesis of cardiomyocytes compared to CM; n = 5 plates/group. D . Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*, and CM* +neu-Ab Hi-FGF-2 . Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase surface area of cardiomyocytes compared to CM; n = 480/group. Please note that for the experiments shown in B,C,D panels the conditioned media in the first two groups (CM, CM*) were supplemented with non-specific rabbit IgG, at 20 µg/ml. E . Representative images of cardiomyocytes stained for anti-N-cadherin (green), alpha-actinin (red) and nuclei (blue), and incubated with CM, CM*, and CM* +neu-Ab (FGF-2). Sizing bar in (iii) coresponds to 100 µM. In all graphs, brackets show comparison between groups, where *, **, ***, ns correspond to P

    Journal: PLoS ONE

    Article Title: High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling

    doi: 10.1371/journal.pone.0097281

    Figure Lengend Snippet: Human Hi-FGF-2 exerts pro-hypertrophic effect. Panel A. Neonatal rat cardiomyocyte cell surface area (normalized, assigning a value of 1 in control, untreated samples) is shown in response to stimulation with Endothelin 1 (ET-1), serving as a positive control, and a recombinant human Hi-FGF-2 preparation (10 ng protein/ml), n = 320 myocytes/group. CM denotes conditioned medium obtained from unstimulated hMFs while CM* denotes conditioned medium from Ang II-stimulated hMFs. ET-1, recombinant human Hi-FGF-2, as well as CM* (but not CM, or Ang II added at 100 nM) increased myocyte cell surface area significantly. Panel B . Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*or CM* supplemented with neutralizing antibodies to total FGF-2 (neu-Ab FGF-2 ), as indicated; n = 480 cells/group. Neutralization of total FGF-2 eliminated the ability of CM* to increase myocytes cell surface area compared to CM. Panel C. Protein synthesis ( 3 H-Leucine incorporation) of cardiomyocytes incubated with CM, CM*, and CM* supplemented with 20 µg/ml neutralizing anti-Hi-FGF-2 antibodies (CM* +neu-Ab Hi-FGF-2 ). Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase protein synthesis of cardiomyocytes compared to CM; n = 5 plates/group. D . Cardiomyocyte cell surface area (normalized) is shown as a function of incubation with CM, CM*, and CM* +neu-Ab Hi-FGF-2 . Neutralization of Hi-FGF-2 eliminated the ability of CM* to increase surface area of cardiomyocytes compared to CM; n = 480/group. Please note that for the experiments shown in B,C,D panels the conditioned media in the first two groups (CM, CM*) were supplemented with non-specific rabbit IgG, at 20 µg/ml. E . Representative images of cardiomyocytes stained for anti-N-cadherin (green), alpha-actinin (red) and nuclei (blue), and incubated with CM, CM*, and CM* +neu-Ab (FGF-2). Sizing bar in (iii) coresponds to 100 µM. In all graphs, brackets show comparison between groups, where *, **, ***, ns correspond to P

    Article Snippet: Isolation of anti-human Hi-FGF-2 antibodies by affinity chromatography Recombinant human Hi-FGF-2 (24 kDa isoform) was cross-linked to CNBr-activated Sepharose (GE Healthcare), as per manufacturer's instructions.

    Techniques: Positive Control, Recombinant, Incubation, Neutralization, Staining

    Detection of Hi-FGF-2 in human atrial tissue. Panel (A) shows representative western blot images of human atrial extracts (hA1, hA2, hA3, 50 µg/lane) probed for FGF-2 with an antibody detecting all FGF-2 isoforms. Expected migration of all human FGF-2 isoforms (34, 24, 22–22.5, and 18 kDa), corresponding to Hi- or Lo-FGF-2, is indicated by arrows; please note that the 34 kDa isoform is not detectable in tissue lysates. Western blots were also probed for cardiac troponin T (TnT) to verify equivalent loading of lanes. Samples hA1, hA2 were analyzed in small (8.3×5.5 cm 2 ) 15% polyacrylamide gels, while hA3 was analyzed in a large (16×11.5 cm 2 ) 15% polyacrylamide gel. The included graph shows percentage of each isoform over total FGF-2, where, n = 45; comparisons between groups are indicated by brackets, where *** and ** denote P

    Journal: PLoS ONE

    Article Title: High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling

    doi: 10.1371/journal.pone.0097281

    Figure Lengend Snippet: Detection of Hi-FGF-2 in human atrial tissue. Panel (A) shows representative western blot images of human atrial extracts (hA1, hA2, hA3, 50 µg/lane) probed for FGF-2 with an antibody detecting all FGF-2 isoforms. Expected migration of all human FGF-2 isoforms (34, 24, 22–22.5, and 18 kDa), corresponding to Hi- or Lo-FGF-2, is indicated by arrows; please note that the 34 kDa isoform is not detectable in tissue lysates. Western blots were also probed for cardiac troponin T (TnT) to verify equivalent loading of lanes. Samples hA1, hA2 were analyzed in small (8.3×5.5 cm 2 ) 15% polyacrylamide gels, while hA3 was analyzed in a large (16×11.5 cm 2 ) 15% polyacrylamide gel. The included graph shows percentage of each isoform over total FGF-2, where, n = 45; comparisons between groups are indicated by brackets, where *** and ** denote P

    Article Snippet: Isolation of anti-human Hi-FGF-2 antibodies by affinity chromatography Recombinant human Hi-FGF-2 (24 kDa isoform) was cross-linked to CNBr-activated Sepharose (GE Healthcare), as per manufacturer's instructions.

    Techniques: Western Blot, Migration

    Detection of Hi-FGF-2 in human atrial myofibroblasts. Panel (A) shows two sets of western blots analyzing FGF-2 isoforms. The first set, (i), is a composite of two blots (separated by a broken line) and analyzes FGF-2 isoforms in hMF lysates (20 µg/lane), from atrial myofibroblast primary cultures obtained from 10 patients (patients 11–20), and correspondingly labeled as C11–20. The second set, (ii), also a composite of two blots separated by a broken line, analyzes FGF-2 isoforms in atrial tissue lysates from patients 11–20, and labelled T11–20 (50 µg/lane). The hMF blots or tissue blots were also probed for, respectively, β-tubulin (β-tub), or Troponin-T (TnT), as indicated. Following densitometry of the hMF blots, the % contribution of each FGF-2 isoform to the total FGF-2 signal was determined for each individual lane, and cumulative results (mean±SD) are included in graph form (n = 10). In Panel (B) , a western blot shows FGF-2 signals from 0.5 and 0.2 ng/lane of recombinant histidine tagged Lo-FGF-2 (FGF-2 His ), atrial tissue lysates (T11, T15 and T17, loaded at 50 µg/lane), side by side with FGF-2 signals from lysates obtained from corresponding primary hMF cultures (C11, C15 and C17, loaded at 10 µg/lane). The graph shows comparisons between tissue and cell lysates for their relative total FGF-2 content, assessed by densitometry as optical density (O.D.) units (n = 3). Measurements corresponding to cell FGF-2 were multiplied by 5, to correct for the 5-fold difference in total protein loading. In both panels, comparisons between groups are indicated by brackets, where P > 0.05 is marked as ns, while P

    Journal: PLoS ONE

    Article Title: High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling

    doi: 10.1371/journal.pone.0097281

    Figure Lengend Snippet: Detection of Hi-FGF-2 in human atrial myofibroblasts. Panel (A) shows two sets of western blots analyzing FGF-2 isoforms. The first set, (i), is a composite of two blots (separated by a broken line) and analyzes FGF-2 isoforms in hMF lysates (20 µg/lane), from atrial myofibroblast primary cultures obtained from 10 patients (patients 11–20), and correspondingly labeled as C11–20. The second set, (ii), also a composite of two blots separated by a broken line, analyzes FGF-2 isoforms in atrial tissue lysates from patients 11–20, and labelled T11–20 (50 µg/lane). The hMF blots or tissue blots were also probed for, respectively, β-tubulin (β-tub), or Troponin-T (TnT), as indicated. Following densitometry of the hMF blots, the % contribution of each FGF-2 isoform to the total FGF-2 signal was determined for each individual lane, and cumulative results (mean±SD) are included in graph form (n = 10). In Panel (B) , a western blot shows FGF-2 signals from 0.5 and 0.2 ng/lane of recombinant histidine tagged Lo-FGF-2 (FGF-2 His ), atrial tissue lysates (T11, T15 and T17, loaded at 50 µg/lane), side by side with FGF-2 signals from lysates obtained from corresponding primary hMF cultures (C11, C15 and C17, loaded at 10 µg/lane). The graph shows comparisons between tissue and cell lysates for their relative total FGF-2 content, assessed by densitometry as optical density (O.D.) units (n = 3). Measurements corresponding to cell FGF-2 were multiplied by 5, to correct for the 5-fold difference in total protein loading. In both panels, comparisons between groups are indicated by brackets, where P > 0.05 is marked as ns, while P

    Article Snippet: Isolation of anti-human Hi-FGF-2 antibodies by affinity chromatography Recombinant human Hi-FGF-2 (24 kDa isoform) was cross-linked to CNBr-activated Sepharose (GE Healthcare), as per manufacturer's instructions.

    Techniques: Western Blot, Labeling, Recombinant

    Selective neutralization of extracellular human Hi-FGF-2 attenuates expression of pro-fibrotic proteins. Panel A . Western blots showing the effect of incubation with either control antibodies (Cont-Ab, 20 µg/ml, lanes 1,2,3), or anti-Hi-FGF-2 antibodies (Neu-Ab Hi-FGF-2 , 20 µg/ml, lanes 4,5,6) on the accumulation of α-SMA, procollagen, SMemb, EDA-Fibronectin (EDA-FN), β-tubulin, and GAPDH, by hMFs, as indicated. Panels B , C , D and E show corresponding quantitative (densitometry) data for α-SMA, procollagen, SMemb, EDA-Fibronectin (EDA-FN), as indicated (±SEM). Incubation with Neu-Ab Hi-FGF-2 significantly decreased expression of α-SMA, procollagen, SMemb and EDA-Fibronectin, without having any effect on GAPDH or β-tubulin. Brackets show comparisons between groups, where *, **, *** correspond to P

    Journal: PLoS ONE

    Article Title: High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling

    doi: 10.1371/journal.pone.0097281

    Figure Lengend Snippet: Selective neutralization of extracellular human Hi-FGF-2 attenuates expression of pro-fibrotic proteins. Panel A . Western blots showing the effect of incubation with either control antibodies (Cont-Ab, 20 µg/ml, lanes 1,2,3), or anti-Hi-FGF-2 antibodies (Neu-Ab Hi-FGF-2 , 20 µg/ml, lanes 4,5,6) on the accumulation of α-SMA, procollagen, SMemb, EDA-Fibronectin (EDA-FN), β-tubulin, and GAPDH, by hMFs, as indicated. Panels B , C , D and E show corresponding quantitative (densitometry) data for α-SMA, procollagen, SMemb, EDA-Fibronectin (EDA-FN), as indicated (±SEM). Incubation with Neu-Ab Hi-FGF-2 significantly decreased expression of α-SMA, procollagen, SMemb and EDA-Fibronectin, without having any effect on GAPDH or β-tubulin. Brackets show comparisons between groups, where *, **, *** correspond to P

    Article Snippet: Isolation of anti-human Hi-FGF-2 antibodies by affinity chromatography Recombinant human Hi-FGF-2 (24 kDa isoform) was cross-linked to CNBr-activated Sepharose (GE Healthcare), as per manufacturer's instructions.

    Techniques: Neutralization, Expressing, Western Blot, Incubation

    Effect of extracellular-acting FGF-2 isoforms on the accumulation of pro-IL-1β and PAI-1 by hMFs. Panel A , western blot and corresponding cumulative data showing relative pro-IL-1β levels (optical density, O.D. units) in hMF cell lysates from unstimulated cells (lanes 1,2,3) and cells stimulated with 10 ng/ml of a recombinant Hi-FGF-2 preparation (Hi, lanes 4,5,6) or 10 ng/ml of recombinant Lo-FGF-2 (Lo, lanes 7,8,9), as indicated. Both Hi- and Lo-FGF-2 upregulated pro-IL-1β, although the effect of Hi-FGF-2 was significantly more potent. Panel B , western blot and corresponding quantitative data showing relative PAI-1 levels (optical density, O.D. units) in hMF cell lysates from unstimulated cells (lanes 1,2,3) and cells stimulated with Hi-FGF-2 (Hi, lanes 4,5,6) or Lo-FGF-2 (Lo, lanes 7,8,9), as indicated. Hi- but not Lo-FGF-2 upregulated PAI-1 levels. Brackets mark comparisons between groups where *, **, ***, and ns denotes P

    Journal: PLoS ONE

    Article Title: High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling

    doi: 10.1371/journal.pone.0097281

    Figure Lengend Snippet: Effect of extracellular-acting FGF-2 isoforms on the accumulation of pro-IL-1β and PAI-1 by hMFs. Panel A , western blot and corresponding cumulative data showing relative pro-IL-1β levels (optical density, O.D. units) in hMF cell lysates from unstimulated cells (lanes 1,2,3) and cells stimulated with 10 ng/ml of a recombinant Hi-FGF-2 preparation (Hi, lanes 4,5,6) or 10 ng/ml of recombinant Lo-FGF-2 (Lo, lanes 7,8,9), as indicated. Both Hi- and Lo-FGF-2 upregulated pro-IL-1β, although the effect of Hi-FGF-2 was significantly more potent. Panel B , western blot and corresponding quantitative data showing relative PAI-1 levels (optical density, O.D. units) in hMF cell lysates from unstimulated cells (lanes 1,2,3) and cells stimulated with Hi-FGF-2 (Hi, lanes 4,5,6) or Lo-FGF-2 (Lo, lanes 7,8,9), as indicated. Hi- but not Lo-FGF-2 upregulated PAI-1 levels. Brackets mark comparisons between groups where *, **, ***, and ns denotes P

    Article Snippet: Isolation of anti-human Hi-FGF-2 antibodies by affinity chromatography Recombinant human Hi-FGF-2 (24 kDa isoform) was cross-linked to CNBr-activated Sepharose (GE Healthcare), as per manufacturer's instructions.

    Techniques: Western Blot, Recombinant

    Detection of Hi-FGF-2 in the extracellular environment in vitro and in vivo . Panel (A) . Representative western blot images of FGF-2 detection in conditioned medium from unstimulated or Ang II-stimulated hMFs. Each lane contains the heparin-sepharose-bound fraction from 60 ml of pooled conditioned medium. Panel (B) . Representative western blots for FGF-2 “eluted” from the cell surface with a high salt wash, and concentrated by binding to heparin-sepharose. Each lane contains the heparin-bound fraction from a 10 ml wash (5×100 near-confluent plates). Ponceau S Red (P-Red) staining of unidentified protein band(s) is also shown, indicative of equivalent loading. Experiments shown in A and B were repeated 2 more times, with similar results. Panel (C) . Western blot image, and corresponding quantitative data of FGF-2 isoforms present in human pericardial fluid (n = 10). Lanes 1-5 (gel 1) and 6-10 (gel 2) contain the heparin-sepharose-bound fraction from 0.5 ml pericardial fluid of individual patients. Lanes marked as F1, F2 contain recombinant Lo-FGF-2 (histidine-tagged) loaded at 0.25 and 0.5 ng/lane respectively. Sample 10 was deliberately overexposed to increase visibility of bands. Recombinant FGF-2, used as standard, was included in the second gel as well (not shown here). The graph shows percent contribution of Hi- or Lo-FGF-2 isoforms to the total FGF-2 signal (mean ± SEM). In all panels, brackets show comparisons between groups; * and ** correspond to P

    Journal: PLoS ONE

    Article Title: High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling

    doi: 10.1371/journal.pone.0097281

    Figure Lengend Snippet: Detection of Hi-FGF-2 in the extracellular environment in vitro and in vivo . Panel (A) . Representative western blot images of FGF-2 detection in conditioned medium from unstimulated or Ang II-stimulated hMFs. Each lane contains the heparin-sepharose-bound fraction from 60 ml of pooled conditioned medium. Panel (B) . Representative western blots for FGF-2 “eluted” from the cell surface with a high salt wash, and concentrated by binding to heparin-sepharose. Each lane contains the heparin-bound fraction from a 10 ml wash (5×100 near-confluent plates). Ponceau S Red (P-Red) staining of unidentified protein band(s) is also shown, indicative of equivalent loading. Experiments shown in A and B were repeated 2 more times, with similar results. Panel (C) . Western blot image, and corresponding quantitative data of FGF-2 isoforms present in human pericardial fluid (n = 10). Lanes 1-5 (gel 1) and 6-10 (gel 2) contain the heparin-sepharose-bound fraction from 0.5 ml pericardial fluid of individual patients. Lanes marked as F1, F2 contain recombinant Lo-FGF-2 (histidine-tagged) loaded at 0.25 and 0.5 ng/lane respectively. Sample 10 was deliberately overexposed to increase visibility of bands. Recombinant FGF-2, used as standard, was included in the second gel as well (not shown here). The graph shows percent contribution of Hi- or Lo-FGF-2 isoforms to the total FGF-2 signal (mean ± SEM). In all panels, brackets show comparisons between groups; * and ** correspond to P

    Article Snippet: Isolation of anti-human Hi-FGF-2 antibodies by affinity chromatography Recombinant human Hi-FGF-2 (24 kDa isoform) was cross-linked to CNBr-activated Sepharose (GE Healthcare), as per manufacturer's instructions.

    Techniques: In Vitro, In Vivo, Western Blot, Binding Assay, Staining, Recombinant

    ERK and MMP-2 activities mediate the Ang II-induced Hi-FGF-2 upregulation in hMFs. Panel A. Western blot and corresponding cumulative data showing the effect of an ERK inhibitor (U0126), or MMP-2 inhibitor (MMP2i) on the Ang II induced Hi-FGF-2 upregulation. Signal for β-tubulin is also shown, serving as loading control. Panel B. Western blots and corresponding cumulative data showing the effect of Ang II administration on phospho-(P)-ERK and total ERK, after 10–30 minutes and 6–24 hours of stimulation as indicated. The graph shows cumulative data (n = 3) of the ratio between P-ERK/ERK over time (10–30 min, 6–24 hours), in response to Ang II. Minutes and hours are indicated as ‘ and h. Panel (C) Representative zymogram of MMP-2 activity in hMFs, including a positive control band (MMP-2), and corresponding cumulative data, showing relative MMP-2 activity in response to Ang II, over time (10–30 min, 6–24 hours), as indicated. For all graphs, brackets show comparisons between groups; *, **, ***, and ns correspond to P

    Journal: PLoS ONE

    Article Title: High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling

    doi: 10.1371/journal.pone.0097281

    Figure Lengend Snippet: ERK and MMP-2 activities mediate the Ang II-induced Hi-FGF-2 upregulation in hMFs. Panel A. Western blot and corresponding cumulative data showing the effect of an ERK inhibitor (U0126), or MMP-2 inhibitor (MMP2i) on the Ang II induced Hi-FGF-2 upregulation. Signal for β-tubulin is also shown, serving as loading control. Panel B. Western blots and corresponding cumulative data showing the effect of Ang II administration on phospho-(P)-ERK and total ERK, after 10–30 minutes and 6–24 hours of stimulation as indicated. The graph shows cumulative data (n = 3) of the ratio between P-ERK/ERK over time (10–30 min, 6–24 hours), in response to Ang II. Minutes and hours are indicated as ‘ and h. Panel (C) Representative zymogram of MMP-2 activity in hMFs, including a positive control band (MMP-2), and corresponding cumulative data, showing relative MMP-2 activity in response to Ang II, over time (10–30 min, 6–24 hours), as indicated. For all graphs, brackets show comparisons between groups; *, **, ***, and ns correspond to P

    Article Snippet: Isolation of anti-human Hi-FGF-2 antibodies by affinity chromatography Recombinant human Hi-FGF-2 (24 kDa isoform) was cross-linked to CNBr-activated Sepharose (GE Healthcare), as per manufacturer's instructions.

    Techniques: Western Blot, Activity Assay, Positive Control

    Both AT-1R and AT-2R mediate the Ang II-induced ERK activation in hMFs. Panel A shows western blot of activated (phosphorylated) pERK, and total ERK, in hMFs stimulated for 30 minutes with with Ang II (lanes 1,2,3), Ang II + PD123319 (lanes 4,5,6), Ang II + Losartan (lanes 7,8,9), and Ang II +PD123319 +Losartan (lanes 10,11,12), in the absence (-) or presence (+) of neutralizing anti-FGF-2 antibodies (neu-Ab FGF-2 ), as indicated. Please note that the western blot for pERK in the groups incubated with neu-Ab FGF-2 is not directly comparable to the western blot for pERK in the groups incubated in the absence of neu-Ab FGF-2 (different exposures). Panel B shows pERK/ERK ratios in the groups shown in panel A. Brackets show statistically significant differences between groups, where *, **, ***, correspond to P

    Journal: PLoS ONE

    Article Title: High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling

    doi: 10.1371/journal.pone.0097281

    Figure Lengend Snippet: Both AT-1R and AT-2R mediate the Ang II-induced ERK activation in hMFs. Panel A shows western blot of activated (phosphorylated) pERK, and total ERK, in hMFs stimulated for 30 minutes with with Ang II (lanes 1,2,3), Ang II + PD123319 (lanes 4,5,6), Ang II + Losartan (lanes 7,8,9), and Ang II +PD123319 +Losartan (lanes 10,11,12), in the absence (-) or presence (+) of neutralizing anti-FGF-2 antibodies (neu-Ab FGF-2 ), as indicated. Please note that the western blot for pERK in the groups incubated with neu-Ab FGF-2 is not directly comparable to the western blot for pERK in the groups incubated in the absence of neu-Ab FGF-2 (different exposures). Panel B shows pERK/ERK ratios in the groups shown in panel A. Brackets show statistically significant differences between groups, where *, **, ***, correspond to P

    Article Snippet: Isolation of anti-human Hi-FGF-2 antibodies by affinity chromatography Recombinant human Hi-FGF-2 (24 kDa isoform) was cross-linked to CNBr-activated Sepharose (GE Healthcare), as per manufacturer's instructions.

    Techniques: Activation Assay, Western Blot, Incubation

    Angiotensin II promotes upregulation of cell-associated human Hi-FGF-2 via AT-1R and AT-2R. Panel A : western blot, and corresponding cumulative data, showing the effect of Ang II on Hi-FGF-2 accumulation by hMFs, in the absence or presence of either losartan (AT-1R inhibitor) or PD123319 (AT-2R inhibitor). Lanes 1–3; 4–6; 7–9; 10–12 correspond to lysates from, respectively, untreated (Control)-;Ang II-stimulated-; Ang II stimulated in the presence of losartan; and Ang II-stimulated in the presence of PD123319- hMFs. Ang II promotes Hi-FGF-2 upregulation which is significantly decreased by either losartan or PD123319. Panel B : western blot and cumulative densitometry data showing the effect of Ang II on Hi-FGF-2 accumulation in the absence or presence of simultaneous inhibition of both AT-1R and AT-2R. Lanes 1–3; 4–6; 7–9 correspond to lysates from, respectively, untreated (Control)-;Ang II-stimulated-; Ang II stimulated in the presence of both losartan and PD123319- hMFs. Relative levels of Hi-FGF-2 in the presence of both AT-1R and AT-2R inhibitors are not significantly different to those of unstimulated cells. Panels C and D . Western blots showing expression, respectively, of AT-1R or AT-2R by hMFs, and relative levels of these receptors after 24 h stimulation with Ang II. After 24 hour stimulation, levels of AT-1R, but not AT-R2, decrease compared to unstimulated cells. Signal for β-actin is also shown in A-D, serving as loading control. E . Densitometry data showing the effect of Ang II receptor inhibitors on baseline Hi-FGF-2 accumulation by hMFs in the abcence of stimulation by added Ang II. Incubation of unstimulated hMFs with losartan (but not PD123319) significantly decreased baseline Hi-FGF-2 levels. Sample size n = 3 (all graphs); *, **, *** indicates P

    Journal: PLoS ONE

    Article Title: High Molecular Weight Fibroblast Growth Factor-2 in the Human Heart Is a Potential Target for Prevention of Cardiac Remodeling

    doi: 10.1371/journal.pone.0097281

    Figure Lengend Snippet: Angiotensin II promotes upregulation of cell-associated human Hi-FGF-2 via AT-1R and AT-2R. Panel A : western blot, and corresponding cumulative data, showing the effect of Ang II on Hi-FGF-2 accumulation by hMFs, in the absence or presence of either losartan (AT-1R inhibitor) or PD123319 (AT-2R inhibitor). Lanes 1–3; 4–6; 7–9; 10–12 correspond to lysates from, respectively, untreated (Control)-;Ang II-stimulated-; Ang II stimulated in the presence of losartan; and Ang II-stimulated in the presence of PD123319- hMFs. Ang II promotes Hi-FGF-2 upregulation which is significantly decreased by either losartan or PD123319. Panel B : western blot and cumulative densitometry data showing the effect of Ang II on Hi-FGF-2 accumulation in the absence or presence of simultaneous inhibition of both AT-1R and AT-2R. Lanes 1–3; 4–6; 7–9 correspond to lysates from, respectively, untreated (Control)-;Ang II-stimulated-; Ang II stimulated in the presence of both losartan and PD123319- hMFs. Relative levels of Hi-FGF-2 in the presence of both AT-1R and AT-2R inhibitors are not significantly different to those of unstimulated cells. Panels C and D . Western blots showing expression, respectively, of AT-1R or AT-2R by hMFs, and relative levels of these receptors after 24 h stimulation with Ang II. After 24 hour stimulation, levels of AT-1R, but not AT-R2, decrease compared to unstimulated cells. Signal for β-actin is also shown in A-D, serving as loading control. E . Densitometry data showing the effect of Ang II receptor inhibitors on baseline Hi-FGF-2 accumulation by hMFs in the abcence of stimulation by added Ang II. Incubation of unstimulated hMFs with losartan (but not PD123319) significantly decreased baseline Hi-FGF-2 levels. Sample size n = 3 (all graphs); *, **, *** indicates P

    Article Snippet: Isolation of anti-human Hi-FGF-2 antibodies by affinity chromatography Recombinant human Hi-FGF-2 (24 kDa isoform) was cross-linked to CNBr-activated Sepharose (GE Healthcare), as per manufacturer's instructions.

    Techniques: Western Blot, Inhibition, Expressing, Incubation