Structured Review

GE Healthcare chromatographies
MALDI-TOF analysis of a rhGCase sample purified by HIC and IEC <t>chromatography.</t> Red bars indicate rhGCase tryptic peptides assigned to human GCase regions. The position of the five putative N-glycosylation sites is shown by circles; all sites except the last one, which never harbours glycans (Berg-Fussman et al. 1993 ), are normally occupied by oligosaccharidic chains.
Chromatographies, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Endosperm-specific expression of human acid beta-glucosidase in a waxy rice"

Article Title: Endosperm-specific expression of human acid beta-glucosidase in a waxy rice

Journal: Rice

doi: 10.1186/1939-8433-5-34

MALDI-TOF analysis of a rhGCase sample purified by HIC and IEC chromatography. Red bars indicate rhGCase tryptic peptides assigned to human GCase regions. The position of the five putative N-glycosylation sites is shown by circles; all sites except the last one, which never harbours glycans (Berg-Fussman et al. 1993 ), are normally occupied by oligosaccharidic chains.
Figure Legend Snippet: MALDI-TOF analysis of a rhGCase sample purified by HIC and IEC chromatography. Red bars indicate rhGCase tryptic peptides assigned to human GCase regions. The position of the five putative N-glycosylation sites is shown by circles; all sites except the last one, which never harbours glycans (Berg-Fussman et al. 1993 ), are normally occupied by oligosaccharidic chains.

Techniques Used: Purification, Hydrophobic Interaction Chromatography, Chromatography

SDS-PAGE analysis carried out to estimate contaminant removal obtained in each chromatographic step. Lane 1: HIC (Hydrophobic Interaction Chromatography) eluate; 2: IEC (Ion Exchange Chromatography) eluate; 3: GF (Gel Filtration) eluate.
Figure Legend Snippet: SDS-PAGE analysis carried out to estimate contaminant removal obtained in each chromatographic step. Lane 1: HIC (Hydrophobic Interaction Chromatography) eluate; 2: IEC (Ion Exchange Chromatography) eluate; 3: GF (Gel Filtration) eluate.

Techniques Used: SDS Page, Hydrophobic Interaction Chromatography, Ion Exchange Chromatography, Filtration

Related Articles

Purification:

Article Title: Endosperm-specific expression of human acid beta-glucosidase in a waxy rice
Article Snippet: .. All chromatographies were performed with the AKTA Prime purification system (GE Healthcare). .. For HIC, two 5-mL HiTrap Octyl FF columns (GE Healthcare) were serially connected and equilibrated with one column volume of loading buffer (10 mL of 200 mM ammonium sulphate in 50 mM Tris-HCl buffer, pH 7.0).

Article Title: Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants
Article Snippet: .. The AdT was purified from the S100 extract by chromatographies on DEAE–cellulose, Phosphocellulose (Whatman), and Hydroxyapatite (Bio-Rad). ..

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    GE Healthcare irc15p size exclusion chromatography
    Redox potential determination of <t>Irc15p</t> in the presence of safranine T. (A) The absorption spectrum of the fully oxidized and fully reduced species are represented by a solid and dashed black line, respectively. Selected spectra of the course of reduction are represented in different shades of blue. 10 μM Irc15p was reduced by the xanthine/xanthine oxidase electron delivering system in the presence of safranine T over a time period of ~100 min. Data points for evaluation were extracted at 430 nm and 530 nm for Irc15p and for the dye safranine T, respectively. (B) Double logarithmic plot of the concentration of oxidized/reduced Irc15p vs. the concentration of oxidized/reduced safranine T (Nernst plot).
    Irc15p Size Exclusion Chromatography, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    GE Healthcare gel filtration chromatography whole cell extracts
    Mrpl44 forms multimers as part of the large subunit of the mitoribosome. (A) HEK293T cells were co-transfected with Mrpl44FLAG and Mrpl44GFP; or FLAGBak and Mrpl44GFP, as a negative control. Lysates were immunoprecipitated with αFLAG, then blotted with an αGFP antibody to determine co-IP. Results are representative of three independent experiments. (B) NIH3T3 <t>whole</t> <t>cell</t> <t>extracts</t> were fractionated by HPLC on a Superose 6 column. The fractions were collected and alternate fractions from 14 to 30 were run on an SDS-PAGE <t>gel,</t> then blotted with antibodies against Mrpl44 as well as Mrpl12 and Mrps15, known components of the mitoribosome. (C) Mitochondrial fractions were obtained from Mrpl44FLAG NIH3T3 cells, and FLAGBak NIH3T3 cells, as a negative control, and immunoprecipitated with αFLAG agarose beads. RNA was then extracted and analysed for pulldown of the mitochondrial rRNA subunits by quantitative RT-PCR. The mean +/- SEM of three independent experiments is shown. Statistical analysis performed using multiple t-test with Holm-Sidak correction for multiple comparisons (*p
    Gel Filtration Chromatography Whole Cell Extracts, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare gel filtration chromatography protein solutions
    <t>Gel-filtration</t> <t>chromatography</t> of Pmr and Pmr-R8A. (A) Elution profiles of Pmr (blue line) and Pmr-R8A (red line). The figures present the absorbance at 280 nm as a function of the elution volume (ml). One milliliter of purified proteins at 140 µM was applied to a HiLoad 16/60 Superdex 200 prep-grade column in buffer B (20 mM Tris-HCl (pH 7.5, 4°C), 0.5 M NaCl, 10% glycerol, and 0.5 M imidazole). The numbers indicate the fractions which were applied to Tricine-SDS-PAGE and Western blot analysis. (B) Tricine-SDS-PAGE profiles of fractions from gel filtration chromatography. The numbers correspond to elution profiles shown in panel A. Ten microliters were applied to Tricine-SDS-PAGE from 1 ml of the fraction. “M” indicates the <t>protein</t> marker. (C) Western blot analysis using anti-His antibody and the same samples in Tricine-SDS-PAGE shown in panel B.
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    Redox potential determination of Irc15p in the presence of safranine T. (A) The absorption spectrum of the fully oxidized and fully reduced species are represented by a solid and dashed black line, respectively. Selected spectra of the course of reduction are represented in different shades of blue. 10 μM Irc15p was reduced by the xanthine/xanthine oxidase electron delivering system in the presence of safranine T over a time period of ~100 min. Data points for evaluation were extracted at 430 nm and 530 nm for Irc15p and for the dye safranine T, respectively. (B) Double logarithmic plot of the concentration of oxidized/reduced Irc15p vs. the concentration of oxidized/reduced safranine T (Nernst plot).

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Redox potential determination of Irc15p in the presence of safranine T. (A) The absorption spectrum of the fully oxidized and fully reduced species are represented by a solid and dashed black line, respectively. Selected spectra of the course of reduction are represented in different shades of blue. 10 μM Irc15p was reduced by the xanthine/xanthine oxidase electron delivering system in the presence of safranine T over a time period of ~100 min. Data points for evaluation were extracted at 430 nm and 530 nm for Irc15p and for the dye safranine T, respectively. (B) Double logarithmic plot of the concentration of oxidized/reduced Irc15p vs. the concentration of oxidized/reduced safranine T (Nernst plot).

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: Concentration Assay

    Overall structural similarity of Irc15p and LPD1p. (A) and (B) Structural superposition of LPD1p (grey, PDB code: 1V59 ) and Irc15p (blue/green). The FAD cofactor is displayed in yellow and NADH is shown in magenta. Close‐up view of the active sites of Irc15p (C) and LPD1p (D). Residues close to the FAD isoalloxazine ring are illustrated as grey sticks for both protomers (Lpd1p) or in colors corresponding to the respective protomer (Irc15p). Figures were prepared with the software PyMOL 25 .

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Overall structural similarity of Irc15p and LPD1p. (A) and (B) Structural superposition of LPD1p (grey, PDB code: 1V59 ) and Irc15p (blue/green). The FAD cofactor is displayed in yellow and NADH is shown in magenta. Close‐up view of the active sites of Irc15p (C) and LPD1p (D). Residues close to the FAD isoalloxazine ring are illustrated as grey sticks for both protomers (Lpd1p) or in colors corresponding to the respective protomer (Irc15p). Figures were prepared with the software PyMOL 25 .

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: Software

    UV/Vis absorption spectroscopy. (A) UV–visible absorption spectrum of Irc15p before (solid line) and after denaturation (dashed line). Denaturation of purified Irc15p was carried out in Buffer B (50 mM HEPES, 50 mM NaCl, 1 mM DTT, pH 7.0) containing 0.2% SDS. (B) Absorption spectra observed during the anaerobic photoreduction of Irc15p in 50 mM HEPES, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.0. The solid black line represents the spectrum before irradiation. The reduction proceeds as indicated by the arrow with the dashed dotted line representing the final spectrum. After reoxidation by dioxygen the protein was partially denatured. The solution was cleared by centrifugation and the spectrum recorded (dashed line).

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: UV/Vis absorption spectroscopy. (A) UV–visible absorption spectrum of Irc15p before (solid line) and after denaturation (dashed line). Denaturation of purified Irc15p was carried out in Buffer B (50 mM HEPES, 50 mM NaCl, 1 mM DTT, pH 7.0) containing 0.2% SDS. (B) Absorption spectra observed during the anaerobic photoreduction of Irc15p in 50 mM HEPES, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.0. The solid black line represents the spectrum before irradiation. The reduction proceeds as indicated by the arrow with the dashed dotted line representing the final spectrum. After reoxidation by dioxygen the protein was partially denatured. The solution was cleared by centrifugation and the spectrum recorded (dashed line).

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: Spectroscopy, Purification, Irradiation, Centrifugation

    Alignment of the Irc15p protein sequence with sequences of LPD from S. cerevisiae , E. coli , S. seoulensis and A. vinelandii . The mitochondrial targeting sequence of Lpd1p is highlighted in red. The amino acid signature near the redox‐active disulfide is highlighted in yellow. The respective sequence in Irc15p is highlighted in green. The catalytic His‐Glu diad is highlighted in blue. Other residues in the active site are highlighted in petrol. Residues involved in structural stabilization are highlighted in purple.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Alignment of the Irc15p protein sequence with sequences of LPD from S. cerevisiae , E. coli , S. seoulensis and A. vinelandii . The mitochondrial targeting sequence of Lpd1p is highlighted in red. The amino acid signature near the redox‐active disulfide is highlighted in yellow. The respective sequence in Irc15p is highlighted in green. The catalytic His‐Glu diad is highlighted in blue. Other residues in the active site are highlighted in petrol. Residues involved in structural stabilization are highlighted in purple.

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: Sequencing

    Pre‐steady‐state kinetics of Irc15p to determine reductive rates for NADH. (A) The rate of reduction was determined under anoxic conditions with the stopped flow device equipped with a diode array detector. At least three independent measurements were performed (error bars are shown as standard deviations). The inset displays selected absorption spectra of the reduction of ~20 μM Irc15p with 375 μM NADH. (B) Absorption change at 450 nm of the reduction of ~20 μM Irc15p with 1250 μM NADH. (C) Absorption change at 450 nm of the reduction of ~20 μM Irc15p with 1000 μM NADPH.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Pre‐steady‐state kinetics of Irc15p to determine reductive rates for NADH. (A) The rate of reduction was determined under anoxic conditions with the stopped flow device equipped with a diode array detector. At least three independent measurements were performed (error bars are shown as standard deviations). The inset displays selected absorption spectra of the reduction of ~20 μM Irc15p with 375 μM NADH. (B) Absorption change at 450 nm of the reduction of ~20 μM Irc15p with 1250 μM NADH. (C) Absorption change at 450 nm of the reduction of ~20 μM Irc15p with 1000 μM NADPH.

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: Flow Cytometry

    Limited proteolysis, hydrogen peroxide formation and SAXS data for Irc15p in the presence and absence of NADH. (A) SDS‐PAGE from the limited proteolysis experiment illustrating the effect of NADH and oxygen on the stability of the protein. Each gel has the marker PageRuler™ prestained protein ladder in Lane 1, the remaining lanes display the samples incubated for 0–16 h. (B) Hydrogen peroxide formation in Irc15p over time (0, 1, and 16 h) and in the presence and absence of NADH. (C) SAXS data comparing the experimental radial density distribution (P(r)) of Irc15p incubated with NADH measured after 0 and 12 h compared with a control sample without NADH.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Limited proteolysis, hydrogen peroxide formation and SAXS data for Irc15p in the presence and absence of NADH. (A) SDS‐PAGE from the limited proteolysis experiment illustrating the effect of NADH and oxygen on the stability of the protein. Each gel has the marker PageRuler™ prestained protein ladder in Lane 1, the remaining lanes display the samples incubated for 0–16 h. (B) Hydrogen peroxide formation in Irc15p over time (0, 1, and 16 h) and in the presence and absence of NADH. (C) SAXS data comparing the experimental radial density distribution (P(r)) of Irc15p incubated with NADH measured after 0 and 12 h compared with a control sample without NADH.

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: SDS Page, Marker, Incubation

    Determination of the purity and molecular mass of Irc15p using SDS‐PAGE and analytical size exclusion chromatography. (A) Determination of purity and subunit molecular mass of Irc15p by SDS‐PAGE after purification by affinity chromatography. Lane 1, PageRuler™ prestained protein ladder (10–180 kDa); Lane 2, protein extract before induction; Lane 3, protein extract after induction of IRC15 ; Lane 4, protein fraction after purification by Ni‐NTA‐sepharose. The subunit molecular mass of Irc15p was estimated to ~55 kDa. (B) Determination of native molecular mass of Irc15p (solid and dotted line display the absorption at 280 nm and 450 nm, respectively) using analytical size exclusion chromatography. The insert shows a plot of the partition coefficient ( K av ) against the logarithm of molecular mass of standard proteins (ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 43 kDa; ribonuclease A, 13.7 kDa). The calculated molecular mass of Irc15p (~ 113 kDa, black circle) indicates that Irc15p is present as a dimer.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

    doi: 10.1002/pro.3517

    Figure Lengend Snippet: Determination of the purity and molecular mass of Irc15p using SDS‐PAGE and analytical size exclusion chromatography. (A) Determination of purity and subunit molecular mass of Irc15p by SDS‐PAGE after purification by affinity chromatography. Lane 1, PageRuler™ prestained protein ladder (10–180 kDa); Lane 2, protein extract before induction; Lane 3, protein extract after induction of IRC15 ; Lane 4, protein fraction after purification by Ni‐NTA‐sepharose. The subunit molecular mass of Irc15p was estimated to ~55 kDa. (B) Determination of native molecular mass of Irc15p (solid and dotted line display the absorption at 280 nm and 450 nm, respectively) using analytical size exclusion chromatography. The insert shows a plot of the partition coefficient ( K av ) against the logarithm of molecular mass of standard proteins (ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 43 kDa; ribonuclease A, 13.7 kDa). The calculated molecular mass of Irc15p (~ 113 kDa, black circle) indicates that Irc15p is present as a dimer.

    Article Snippet: To determine the native molecular mass of Irc15p size exclusion chromatography with Buffer A using a Superdex 200 10/300 GL column attached to an Äktapurifier™ system from GE Healthcare (Little Chalfont, UK) was performed.

    Techniques: SDS Page, Size-exclusion Chromatography, Purification, Affinity Chromatography

    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

    Mrpl44 forms multimers as part of the large subunit of the mitoribosome. (A) HEK293T cells were co-transfected with Mrpl44FLAG and Mrpl44GFP; or FLAGBak and Mrpl44GFP, as a negative control. Lysates were immunoprecipitated with αFLAG, then blotted with an αGFP antibody to determine co-IP. Results are representative of three independent experiments. (B) NIH3T3 whole cell extracts were fractionated by HPLC on a Superose 6 column. The fractions were collected and alternate fractions from 14 to 30 were run on an SDS-PAGE gel, then blotted with antibodies against Mrpl44 as well as Mrpl12 and Mrps15, known components of the mitoribosome. (C) Mitochondrial fractions were obtained from Mrpl44FLAG NIH3T3 cells, and FLAGBak NIH3T3 cells, as a negative control, and immunoprecipitated with αFLAG agarose beads. RNA was then extracted and analysed for pulldown of the mitochondrial rRNA subunits by quantitative RT-PCR. The mean +/- SEM of three independent experiments is shown. Statistical analysis performed using multiple t-test with Holm-Sidak correction for multiple comparisons (*p

    Journal: PLoS ONE

    Article Title: A Role for the Mitochondrial Protein Mrpl44 in Maintaining OXPHOS Capacity

    doi: 10.1371/journal.pone.0134326

    Figure Lengend Snippet: Mrpl44 forms multimers as part of the large subunit of the mitoribosome. (A) HEK293T cells were co-transfected with Mrpl44FLAG and Mrpl44GFP; or FLAGBak and Mrpl44GFP, as a negative control. Lysates were immunoprecipitated with αFLAG, then blotted with an αGFP antibody to determine co-IP. Results are representative of three independent experiments. (B) NIH3T3 whole cell extracts were fractionated by HPLC on a Superose 6 column. The fractions were collected and alternate fractions from 14 to 30 were run on an SDS-PAGE gel, then blotted with antibodies against Mrpl44 as well as Mrpl12 and Mrps15, known components of the mitoribosome. (C) Mitochondrial fractions were obtained from Mrpl44FLAG NIH3T3 cells, and FLAGBak NIH3T3 cells, as a negative control, and immunoprecipitated with αFLAG agarose beads. RNA was then extracted and analysed for pulldown of the mitochondrial rRNA subunits by quantitative RT-PCR. The mean +/- SEM of three independent experiments is shown. Statistical analysis performed using multiple t-test with Holm-Sidak correction for multiple comparisons (*p

    Article Snippet: Gel Filtration Chromatography Whole cell extracts were fractionated using a Waters HPLC system on a pre-packed Superose 6 gel-filtration column (300mm x 10mm internal diameter, GE Healthcare) equilibrated in 0.5% Triton X-100 in 20mM Tris-HCl, pH7.5, 150mM NaCl containing protease inhibitors.

    Techniques: Transfection, Negative Control, Immunoprecipitation, Co-Immunoprecipitation Assay, High Performance Liquid Chromatography, SDS Page, Quantitative RT-PCR

    Gel-filtration chromatography of Pmr and Pmr-R8A. (A) Elution profiles of Pmr (blue line) and Pmr-R8A (red line). The figures present the absorbance at 280 nm as a function of the elution volume (ml). One milliliter of purified proteins at 140 µM was applied to a HiLoad 16/60 Superdex 200 prep-grade column in buffer B (20 mM Tris-HCl (pH 7.5, 4°C), 0.5 M NaCl, 10% glycerol, and 0.5 M imidazole). The numbers indicate the fractions which were applied to Tricine-SDS-PAGE and Western blot analysis. (B) Tricine-SDS-PAGE profiles of fractions from gel filtration chromatography. The numbers correspond to elution profiles shown in panel A. Ten microliters were applied to Tricine-SDS-PAGE from 1 ml of the fraction. “M” indicates the protein marker. (C) Western blot analysis using anti-His antibody and the same samples in Tricine-SDS-PAGE shown in panel B.

    Journal: PLoS ONE

    Article Title: Oligomerization Mechanisms of an H-NS Family Protein, Pmr, Encoded on the Plasmid pCAR1 Provide a Molecular Basis for Functions of H-NS Family Members

    doi: 10.1371/journal.pone.0105656

    Figure Lengend Snippet: Gel-filtration chromatography of Pmr and Pmr-R8A. (A) Elution profiles of Pmr (blue line) and Pmr-R8A (red line). The figures present the absorbance at 280 nm as a function of the elution volume (ml). One milliliter of purified proteins at 140 µM was applied to a HiLoad 16/60 Superdex 200 prep-grade column in buffer B (20 mM Tris-HCl (pH 7.5, 4°C), 0.5 M NaCl, 10% glycerol, and 0.5 M imidazole). The numbers indicate the fractions which were applied to Tricine-SDS-PAGE and Western blot analysis. (B) Tricine-SDS-PAGE profiles of fractions from gel filtration chromatography. The numbers correspond to elution profiles shown in panel A. Ten microliters were applied to Tricine-SDS-PAGE from 1 ml of the fraction. “M” indicates the protein marker. (C) Western blot analysis using anti-His antibody and the same samples in Tricine-SDS-PAGE shown in panel B.

    Article Snippet: Gel-filtration chromatography Protein solutions after purification using HiTrap Chelating HP column were applied to a HiLoad 16/60 Superdex 200 prep-grade column (GE Healthcare).

    Techniques: Filtration, Chromatography, Purification, SDS Page, Western Blot, Marker