anti mouse igg whole molecule peroxidase antibody  (Millipore)

 
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    Structured Review

    Millipore anti mouse igg whole molecule peroxidase antibody
    Anti Mouse Igg Whole Molecule Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse igg whole molecule peroxidase antibody/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti mouse igg whole molecule peroxidase antibody - by Bioz Stars, 2021-04
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    Related Articles

    Incubation:

    Article Title: Inhibition of PbGP43 Expression May Suggest that gp43 is a Virulence Factor in Paracoccidioides brasiliensis
    Article Snippet: Blotted proteins were assayed for laminin and fibronectin affinity as follows: the membranes were incubated with 1% bovine serum albumin (BSA) in PBS, pH 7.4, for 4 h at room temperature and then for 90 min in PBS-T-BSA (PBS, 0,05% Tween 20, 5% non fat milk) containing 30 µg/ml of either laminin (derived from Engelbreth-Holm-Swarm murine sarcoma from Sigma) or human fibronectin (Sigma). .. Membranes were washed three times in PBS-T, 10 min each, and then incubated for 1 h under agitation with rabbit anti-mouse laminin or rabbit anti-human fibronectin antibodies (both from Sigma, 1∶100) in PBS-T-BSA under continuous agitation. .. The blots were washed in PBS-T and incubated with peroxidase-labeled goat anti-rabbit immunoglobulin at 1∶1,000 in PBS-T-BSA at room temperature.

    Article Title: The PDZ Protein GIPC Regulates Trafficking of the LPA1 Receptor from APPL Signaling Endosomes and Attenuates the Cell's Response to LPA
    Article Snippet: Insoluble material was removed by centrifugation (10,000×g for 30 min at 4°C), and the protein concentration of the supernatant was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA). .. Cell lysates (3–4 mg protein) were incubated at 4°C with mouse anti-FLAG IgG overnight followed by incubation with protein G-Sepharose beads (Sigma-Aldrich) for 1 h. Beads were then washed extensively with lysis buffer and resuspended in Laemmli sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue) for SDS-PAGE. .. Immunoblotting Proteins separated by SDS-PAGE were transferred to PVDF membranes (Millipore, Billerica, MA).

    Article Title: Two RSV Platforms for G, F, or G+F Proteins VLPs
    Article Snippet: F, Ga, and Gb Antibody EIAs The secreted form of F, Ga, or Gb protein antigens was produced from stably-transfected 293F cells in serum-free media and coated onto a 96-well microtiter plate in coating buffer (15 mM Na2 CO3 , 35 mM NaHCO3 , 3 mM NaN3 , pH 9.4–9.6). .. The plates were incubated in 2% nonfat dry milk dissolved in PBS blocking solution for 2 h at 37 °C, washed with PBS-T (PBS + 0.15% Tween-20), and serum specimens at 1:200 dilution added to the wells, incubated for 1 h at 37 °C, the plates washed with PBS-T, and goat anti mouse IgG-HRP (1:5000) added and incubated for 1 h at 37 °C. .. Color was developed with o -Phenylenediamine dihydrochloride (OPD) (Sigma-Aldrich, St. Louis, MO, USA) substrate and the reaction stopped after 30 min at RT with 4N H2 SO4 .

    Lysis:

    Article Title: The PDZ Protein GIPC Regulates Trafficking of the LPA1 Receptor from APPL Signaling Endosomes and Attenuates the Cell's Response to LPA
    Article Snippet: Insoluble material was removed by centrifugation (10,000×g for 30 min at 4°C), and the protein concentration of the supernatant was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA). .. Cell lysates (3–4 mg protein) were incubated at 4°C with mouse anti-FLAG IgG overnight followed by incubation with protein G-Sepharose beads (Sigma-Aldrich) for 1 h. Beads were then washed extensively with lysis buffer and resuspended in Laemmli sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue) for SDS-PAGE. .. Immunoblotting Proteins separated by SDS-PAGE were transferred to PVDF membranes (Millipore, Billerica, MA).

    SDS Page:

    Article Title: The PDZ Protein GIPC Regulates Trafficking of the LPA1 Receptor from APPL Signaling Endosomes and Attenuates the Cell's Response to LPA
    Article Snippet: Insoluble material was removed by centrifugation (10,000×g for 30 min at 4°C), and the protein concentration of the supernatant was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA). .. Cell lysates (3–4 mg protein) were incubated at 4°C with mouse anti-FLAG IgG overnight followed by incubation with protein G-Sepharose beads (Sigma-Aldrich) for 1 h. Beads were then washed extensively with lysis buffer and resuspended in Laemmli sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue) for SDS-PAGE. .. Immunoblotting Proteins separated by SDS-PAGE were transferred to PVDF membranes (Millipore, Billerica, MA).

    FLAG-tag:

    Article Title: Protein Interaction Analysis of Senataxin and the ALS4 L389S Mutant Yields Insights into Senataxin Post-Translational Modification and Uncovers Mutant-Specific Binding with a Brain Cytoplasmic RNA-Encoded Peptide
    Article Snippet: Protein lysates were resolved using NuPAGE® Tris-Acetate gradient Gels (Invitrogen) and transferred to PVDF membrane (Sigma) by 1 hr of electroblot at 30 volts and blocked with 4% non-fat dry milk. .. To detect FLAG epitope, anti-FLAG M2 antibody (Sigma: F1804) was used (1∶5,000) in conjunction with anti-mouse HRP secondary antibodies (Sigma: A9044) (1∶10,000). .. The ECL Plus HRP detection kit (Amersham) was used for chemiluminescent detection.

    Western Blot:

    Article Title: Decorin-Mediated Regulation of Fibrillin-1 in the Kidney Involves the Insulin-Like Growth Factor-I Receptor and Mammalian Target of Rapamycin
    Article Snippet: TRIzol, reagents for reverse transcriptase-polymerase chain reaction (RT-PCR), cell culture media, and sera were obtained from Invitrogen (Groningen, The Netherlands), and tissue culture plastic was from Falcon (Becton-Dickinson, Heidelberg, Germany). .. Antibodies used for Western blots were as follows: rabbit anti-human fibrillin-1, rabbit-anti-human DCN, rabbit anti-mouse DCN (LF-113), and rabbit anti-human fibronectin (Sigma-Aldrich, Deisenhofen, Germany); mouse antibody to phos-photyrosine (clone PY20; Biomol, Hamburg, Germany); rabbit anti-β-chain of the IGF-IR (sc-713) and rabbit anti-β-tubulin (both from Santa Cruz Biotechnologies, Heidelberg, Germany); and anti-phospho-IGF-R (Tyr1131)/insulin receptor (Tyr1146), anti-phospho-PDK1 (Ser241), anti-PDK1, anti-phospho-PKCζ/λ (Thr410/403), anti-PKCζ, anti-phospho-Akt (Thr 308), anti-phospho-Akt (Ser473), anti-Akt, anti-phosphorylated Erk1/2 (Thr202/204), anti-total-Erk1/2, anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-p70 S6 kinase (Thr389), anti-p70 S6 kinase, anti-phospho-S6 ribosomal protein (Ser235/236), anti-S6 ribosomal protein, anti-phospho-eIF4B (Ser422), and anti-eIF4B (all raised in rabbits and all from Cell Signaling Technologies, Frankfurt/Main, Germany). ..

    Produced:

    Article Title: Evidence of a Cell Surface Role for Hsp90 Complex Proteins Mediating Neuroblast Migration in the Subventricular Zone
    Article Snippet: In both groups, medium was replaced, however in the experimental groups, polyclonal antibodies recognizing Hsp90 (1 mg/mL, dilution 150 μg/mL-Milipore) and HOP/STI1 (2.2 μg/μL, Bethyl, rabbit 33, dilution 1:200; 0.011 μg/μL), as well as Hsp90 inhibitor Geldanamycin (100, 10, and 1 nM-Tocris Bioscience, 1368) and VER155008 (100 nM, 10 nM and 1 nM-Tocris Bioscience, 3803) were added to the medium. .. As a control, SVZ explants were treated anti-mouse IgG produced in Rabbit (Sigma-Aldrich A9044). .. SVZ migration

    Blocking Assay:

    Article Title: Two RSV Platforms for G, F, or G+F Proteins VLPs
    Article Snippet: F, Ga, and Gb Antibody EIAs The secreted form of F, Ga, or Gb protein antigens was produced from stably-transfected 293F cells in serum-free media and coated onto a 96-well microtiter plate in coating buffer (15 mM Na2 CO3 , 35 mM NaHCO3 , 3 mM NaN3 , pH 9.4–9.6). .. The plates were incubated in 2% nonfat dry milk dissolved in PBS blocking solution for 2 h at 37 °C, washed with PBS-T (PBS + 0.15% Tween-20), and serum specimens at 1:200 dilution added to the wells, incubated for 1 h at 37 °C, the plates washed with PBS-T, and goat anti mouse IgG-HRP (1:5000) added and incubated for 1 h at 37 °C. .. Color was developed with o -Phenylenediamine dihydrochloride (OPD) (Sigma-Aldrich, St. Louis, MO, USA) substrate and the reaction stopped after 30 min at RT with 4N H2 SO4 .

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    Millipore rabbit anti mouse dcn
    Schematic diagram of the <t>DCN-mediated</t> translational regulation of <t>fibrillin-1</t> in NRK cells. Further details are given in Results.
    Rabbit Anti Mouse Dcn, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti mouse dcn/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti mouse dcn - by Bioz Stars, 2021-04
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      Buy from Supplier

    99
    Millipore goat anti mouse hrp conjugated polyclonal antibodies
    Sandwich ELISA for detection of BoNT/B. MAb MCS6-27 was used as capture antibody, rabbit anti-BoNT/B <t>polyclonal</t> (Metabiologics) was used in conjunction with an <t>HRP-conjugated,</t> goat anti-rabbit polyclonal antibody as detector. Data are presented ±1 standard error, n = 3. Horizontal dashed lines represent the limit of detection (L.O.D., zero toxin plus 3 standard deviations) and the limit of quantitation (L.O.Q., zero toxin plus 5 standard deviations).
    Goat Anti Mouse Hrp Conjugated Polyclonal Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti mouse hrp conjugated polyclonal antibodies/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti mouse hrp conjugated polyclonal antibodies - by Bioz Stars, 2021-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    Schematic diagram of the DCN-mediated translational regulation of fibrillin-1 in NRK cells. Further details are given in Results.

    Journal:

    Article Title: Decorin-Mediated Regulation of Fibrillin-1 in the Kidney Involves the Insulin-Like Growth Factor-I Receptor and Mammalian Target of Rapamycin

    doi: 10.2353/ajpath.2007.060497

    Figure Lengend Snippet: Schematic diagram of the DCN-mediated translational regulation of fibrillin-1 in NRK cells. Further details are given in Results.

    Article Snippet: Antibodies used for Western blots were as follows: rabbit anti-human fibrillin-1, rabbit-anti-human DCN, rabbit anti-mouse DCN (LF-113), and rabbit anti-human fibronectin (Sigma-Aldrich, Deisenhofen, Germany); mouse antibody to phos-photyrosine (clone PY20; Biomol, Hamburg, Germany); rabbit anti-β-chain of the IGF-IR (sc-713) and rabbit anti-β-tubulin (both from Santa Cruz Biotechnologies, Heidelberg, Germany); and anti-phospho-IGF-R (Tyr1131)/insulin receptor (Tyr1146), anti-phospho-PDK1 (Ser241), anti-PDK1, anti-phospho-PKCζ/λ (Thr410/403), anti-PKCζ, anti-phospho-Akt (Thr 308), anti-phospho-Akt (Ser473), anti-Akt, anti-phosphorylated Erk1/2 (Thr202/204), anti-total-Erk1/2, anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-p70 S6 kinase (Thr389), anti-p70 S6 kinase, anti-phospho-S6 ribosomal protein (Ser235/236), anti-S6 ribosomal protein, anti-phospho-eIF4B (Ser422), and anti-eIF4B (all raised in rabbits and all from Cell Signaling Technologies, Frankfurt/Main, Germany).

    Techniques:

    Decorin and IGF-I induce fibrillin-1 protein synthesis in normal rat kidney fibroblasts via the IGF-IR. A and B show representative Western blots for fibrillin-1 in culture supernatants from NRK cells incubated for 6 hours with recombinant DCN or IGF-I

    Journal:

    Article Title: Decorin-Mediated Regulation of Fibrillin-1 in the Kidney Involves the Insulin-Like Growth Factor-I Receptor and Mammalian Target of Rapamycin

    doi: 10.2353/ajpath.2007.060497

    Figure Lengend Snippet: Decorin and IGF-I induce fibrillin-1 protein synthesis in normal rat kidney fibroblasts via the IGF-IR. A and B show representative Western blots for fibrillin-1 in culture supernatants from NRK cells incubated for 6 hours with recombinant DCN or IGF-I

    Article Snippet: Antibodies used for Western blots were as follows: rabbit anti-human fibrillin-1, rabbit-anti-human DCN, rabbit anti-mouse DCN (LF-113), and rabbit anti-human fibronectin (Sigma-Aldrich, Deisenhofen, Germany); mouse antibody to phos-photyrosine (clone PY20; Biomol, Hamburg, Germany); rabbit anti-β-chain of the IGF-IR (sc-713) and rabbit anti-β-tubulin (both from Santa Cruz Biotechnologies, Heidelberg, Germany); and anti-phospho-IGF-R (Tyr1131)/insulin receptor (Tyr1146), anti-phospho-PDK1 (Ser241), anti-PDK1, anti-phospho-PKCζ/λ (Thr410/403), anti-PKCζ, anti-phospho-Akt (Thr 308), anti-phospho-Akt (Ser473), anti-Akt, anti-phosphorylated Erk1/2 (Thr202/204), anti-total-Erk1/2, anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-p70 S6 kinase (Thr389), anti-p70 S6 kinase, anti-phospho-S6 ribosomal protein (Ser235/236), anti-S6 ribosomal protein, anti-phospho-eIF4B (Ser422), and anti-eIF4B (all raised in rabbits and all from Cell Signaling Technologies, Frankfurt/Main, Germany).

    Techniques: Western Blot, Incubation, Recombinant

    DCN- and IGF-I-dependent regulation of fibrillin-1 in NRK cells involves mTOR. A- - C: DCN- and IGF-I-mediated phosphorylation of mTOR in NRK cells (8 minutes of incubation) was suppressed by inhibition of IGF-IR ( A ), by an inhibition of PI3K ( B ), and by

    Journal:

    Article Title: Decorin-Mediated Regulation of Fibrillin-1 in the Kidney Involves the Insulin-Like Growth Factor-I Receptor and Mammalian Target of Rapamycin

    doi: 10.2353/ajpath.2007.060497

    Figure Lengend Snippet: DCN- and IGF-I-dependent regulation of fibrillin-1 in NRK cells involves mTOR. A- - C: DCN- and IGF-I-mediated phosphorylation of mTOR in NRK cells (8 minutes of incubation) was suppressed by inhibition of IGF-IR ( A ), by an inhibition of PI3K ( B ), and by

    Article Snippet: Antibodies used for Western blots were as follows: rabbit anti-human fibrillin-1, rabbit-anti-human DCN, rabbit anti-mouse DCN (LF-113), and rabbit anti-human fibronectin (Sigma-Aldrich, Deisenhofen, Germany); mouse antibody to phos-photyrosine (clone PY20; Biomol, Hamburg, Germany); rabbit anti-β-chain of the IGF-IR (sc-713) and rabbit anti-β-tubulin (both from Santa Cruz Biotechnologies, Heidelberg, Germany); and anti-phospho-IGF-R (Tyr1131)/insulin receptor (Tyr1146), anti-phospho-PDK1 (Ser241), anti-PDK1, anti-phospho-PKCζ/λ (Thr410/403), anti-PKCζ, anti-phospho-Akt (Thr 308), anti-phospho-Akt (Ser473), anti-Akt, anti-phosphorylated Erk1/2 (Thr202/204), anti-total-Erk1/2, anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-p70 S6 kinase (Thr389), anti-p70 S6 kinase, anti-phospho-S6 ribosomal protein (Ser235/236), anti-S6 ribosomal protein, anti-phospho-eIF4B (Ser422), and anti-eIF4B (all raised in rabbits and all from Cell Signaling Technologies, Frankfurt/Main, Germany).

    Techniques: Incubation, Inhibition

    Involvement of the PI3K/Akt pathway in DCN- and IGF-I-mediated regulation of fibrillin-1 in NRK cells. A: Western blot for fibrillin-1 in culture supernatants from NRK cells incubated for 6 hours with recombinant DCN or IGF-I in the presence or absence

    Journal:

    Article Title: Decorin-Mediated Regulation of Fibrillin-1 in the Kidney Involves the Insulin-Like Growth Factor-I Receptor and Mammalian Target of Rapamycin

    doi: 10.2353/ajpath.2007.060497

    Figure Lengend Snippet: Involvement of the PI3K/Akt pathway in DCN- and IGF-I-mediated regulation of fibrillin-1 in NRK cells. A: Western blot for fibrillin-1 in culture supernatants from NRK cells incubated for 6 hours with recombinant DCN or IGF-I in the presence or absence

    Article Snippet: Antibodies used for Western blots were as follows: rabbit anti-human fibrillin-1, rabbit-anti-human DCN, rabbit anti-mouse DCN (LF-113), and rabbit anti-human fibronectin (Sigma-Aldrich, Deisenhofen, Germany); mouse antibody to phos-photyrosine (clone PY20; Biomol, Hamburg, Germany); rabbit anti-β-chain of the IGF-IR (sc-713) and rabbit anti-β-tubulin (both from Santa Cruz Biotechnologies, Heidelberg, Germany); and anti-phospho-IGF-R (Tyr1131)/insulin receptor (Tyr1146), anti-phospho-PDK1 (Ser241), anti-PDK1, anti-phospho-PKCζ/λ (Thr410/403), anti-PKCζ, anti-phospho-Akt (Thr 308), anti-phospho-Akt (Ser473), anti-Akt, anti-phosphorylated Erk1/2 (Thr202/204), anti-total-Erk1/2, anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-p70 S6 kinase (Thr389), anti-p70 S6 kinase, anti-phospho-S6 ribosomal protein (Ser235/236), anti-S6 ribosomal protein, anti-phospho-eIF4B (Ser422), and anti-eIF4B (all raised in rabbits and all from Cell Signaling Technologies, Frankfurt/Main, Germany).

    Techniques: Western Blot, Incubation, Recombinant

    Decorin deficiency in STZ-induced diabetes is associated with lower accumulation of fibrillin-1 and overexpression of IGF-IR. A and C: Western blots for fibrillin-1 ( A ), IGF-IR ( C ), and β-tubulin in homogenates of diabetic kidneys from Dcn −/−

    Journal:

    Article Title: Decorin-Mediated Regulation of Fibrillin-1 in the Kidney Involves the Insulin-Like Growth Factor-I Receptor and Mammalian Target of Rapamycin

    doi: 10.2353/ajpath.2007.060497

    Figure Lengend Snippet: Decorin deficiency in STZ-induced diabetes is associated with lower accumulation of fibrillin-1 and overexpression of IGF-IR. A and C: Western blots for fibrillin-1 ( A ), IGF-IR ( C ), and β-tubulin in homogenates of diabetic kidneys from Dcn −/−

    Article Snippet: Antibodies used for Western blots were as follows: rabbit anti-human fibrillin-1, rabbit-anti-human DCN, rabbit anti-mouse DCN (LF-113), and rabbit anti-human fibronectin (Sigma-Aldrich, Deisenhofen, Germany); mouse antibody to phos-photyrosine (clone PY20; Biomol, Hamburg, Germany); rabbit anti-β-chain of the IGF-IR (sc-713) and rabbit anti-β-tubulin (both from Santa Cruz Biotechnologies, Heidelberg, Germany); and anti-phospho-IGF-R (Tyr1131)/insulin receptor (Tyr1146), anti-phospho-PDK1 (Ser241), anti-PDK1, anti-phospho-PKCζ/λ (Thr410/403), anti-PKCζ, anti-phospho-Akt (Thr 308), anti-phospho-Akt (Ser473), anti-Akt, anti-phosphorylated Erk1/2 (Thr202/204), anti-total-Erk1/2, anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-p70 S6 kinase (Thr389), anti-p70 S6 kinase, anti-phospho-S6 ribosomal protein (Ser235/236), anti-S6 ribosomal protein, anti-phospho-eIF4B (Ser422), and anti-eIF4B (all raised in rabbits and all from Cell Signaling Technologies, Frankfurt/Main, Germany).

    Techniques: Over Expression, Western Blot

    Trafficking of LPA 1 and GIPC to Endosomes. A, Upper panel : In serum starved cells stably expressing LPA 1 (asterisks), GIPC is concentrated at the plasma membrane (arrowheads) where it colocalizes with LPA 1 . Middle panels : 2 min following stimulation with LPA, LPA 1 colocalizes with APPL and GIPC (arrowheads) in endocytic vesicles at the cell periphery. Lower panel : 30 min following stimulation, LPA 1 colocalizes with EEA1 in early endosomes concentrated in the juxtanuclear region (arrowheads). Boxed regions are enlarged (2.2×) in the insets. Bar = 10 µm. HEK- LPA 1 cells were serum starved for 4 h, incubated on ice with mouse anti-FLAG IgG to label FLAG-LPA 1 at the cell surface, washed in PBS and shifted to fresh medium containing LPA (10 µM) 2 or 5 min before fixation. Cells were processed for immunofluorescence using affinity purified rabbit anti-GIPC, anti-APPL1 or anti-EEA1 IgG, followed by goat anti-rabbit Alexa-488 and goat-anti-mouse Alexa-594 F(ab’) 2 (the latter to detect FLAG-LPA 1 ). Images were taken with a PerkinElmer UltraView Vox Spinning Disk Confocal unit connected to an Olympus IX81 inverted microscope and a EMCCD camera (Hamamatsu) using a 60X oil immersion lens (1.42 NA). B, GIPC co-immunoprecipitates with FLAG-LPA 1 from serum-starved cells (lane 7) at all time points after LPA stimulation, but the interaction gradually decreases after LPA stimulation (lanes 8–10). The relative abundance of GIPC that coprecipitated with FLAG-LPA 1 is indicated beneath each band. As expected, both LPA 1 and GIPC are absent from immunoprecipitates of cells transiently transfected with empty vector instead of FLAG- LPA 1 (lane 6, vector control). HEK293 cells were transiently co-transfected with full-length GIPC and FLAG-LPA 1 (lanes 2–5 and 7–10) or GIPC alone (lanes 1 and 6). Cells were serum starved overnight (lanes 1, 2, 6 and 7) or starved and stimulated with 10 µM LPA for 5 (lanes 3 and 8), 15 (lanes 4 and 9) or 30 min (lanes 5 and 10) before lysis. IP was carried out on cell lysates using mouse anti-FLAG IgG and immunoblotted as is Fig. 1A. The abundance of GIPC and LPA 1 in each immunoprecipitation reaction was quantified using the LICOR imaging system, and the GIPC abundance relative to LPA 1 was calculated for each reaction. Similar results were obtained in 2 additional experiments. Input (lanes 1–5): Lysates (2%) are shown to verify comparable expression levels.

    Journal: PLoS ONE

    Article Title: The PDZ Protein GIPC Regulates Trafficking of the LPA1 Receptor from APPL Signaling Endosomes and Attenuates the Cell's Response to LPA

    doi: 10.1371/journal.pone.0049227

    Figure Lengend Snippet: Trafficking of LPA 1 and GIPC to Endosomes. A, Upper panel : In serum starved cells stably expressing LPA 1 (asterisks), GIPC is concentrated at the plasma membrane (arrowheads) where it colocalizes with LPA 1 . Middle panels : 2 min following stimulation with LPA, LPA 1 colocalizes with APPL and GIPC (arrowheads) in endocytic vesicles at the cell periphery. Lower panel : 30 min following stimulation, LPA 1 colocalizes with EEA1 in early endosomes concentrated in the juxtanuclear region (arrowheads). Boxed regions are enlarged (2.2×) in the insets. Bar = 10 µm. HEK- LPA 1 cells were serum starved for 4 h, incubated on ice with mouse anti-FLAG IgG to label FLAG-LPA 1 at the cell surface, washed in PBS and shifted to fresh medium containing LPA (10 µM) 2 or 5 min before fixation. Cells were processed for immunofluorescence using affinity purified rabbit anti-GIPC, anti-APPL1 or anti-EEA1 IgG, followed by goat anti-rabbit Alexa-488 and goat-anti-mouse Alexa-594 F(ab’) 2 (the latter to detect FLAG-LPA 1 ). Images were taken with a PerkinElmer UltraView Vox Spinning Disk Confocal unit connected to an Olympus IX81 inverted microscope and a EMCCD camera (Hamamatsu) using a 60X oil immersion lens (1.42 NA). B, GIPC co-immunoprecipitates with FLAG-LPA 1 from serum-starved cells (lane 7) at all time points after LPA stimulation, but the interaction gradually decreases after LPA stimulation (lanes 8–10). The relative abundance of GIPC that coprecipitated with FLAG-LPA 1 is indicated beneath each band. As expected, both LPA 1 and GIPC are absent from immunoprecipitates of cells transiently transfected with empty vector instead of FLAG- LPA 1 (lane 6, vector control). HEK293 cells were transiently co-transfected with full-length GIPC and FLAG-LPA 1 (lanes 2–5 and 7–10) or GIPC alone (lanes 1 and 6). Cells were serum starved overnight (lanes 1, 2, 6 and 7) or starved and stimulated with 10 µM LPA for 5 (lanes 3 and 8), 15 (lanes 4 and 9) or 30 min (lanes 5 and 10) before lysis. IP was carried out on cell lysates using mouse anti-FLAG IgG and immunoblotted as is Fig. 1A. The abundance of GIPC and LPA 1 in each immunoprecipitation reaction was quantified using the LICOR imaging system, and the GIPC abundance relative to LPA 1 was calculated for each reaction. Similar results were obtained in 2 additional experiments. Input (lanes 1–5): Lysates (2%) are shown to verify comparable expression levels.

    Article Snippet: Cell lysates (3–4 mg protein) were incubated at 4°C with mouse anti-FLAG IgG overnight followed by incubation with protein G-Sepharose beads (Sigma-Aldrich) for 1 h. Beads were then washed extensively with lysis buffer and resuspended in Laemmli sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue) for SDS-PAGE.

    Techniques: Stable Transfection, Expressing, Incubation, Immunofluorescence, Affinity Purification, Inverted Microscopy, Transfection, Plasmid Preparation, Lysis, Immunoprecipitation, Imaging

    GIPC directly interacts with the C-terminal PDZ binding motif of LPA 1 but not with other LPA receptors. A, Endogenous GIPC and GIPC-GFP co-immunoprecipitate with FLAG- LPA 1 from HEK cells expressing FLAG- LPA 1 (arrowhead, lane 4) but not control HEK cells (lane 3). C-terminally tagged GIPC-GFP and N-terminally tagged FLAG-LPA 1 were transiently coexpressed in HEK293 cells, and immunoprecipitation was carried out on cell lysates with mouse anti-FLAG IgG followed by immunoblotting with mouse anti-FLAG (LPA 1 ) and rabbit anti-GIPC IgG. Lanes were cropped from a single exposure of a continuous membrane. The lower panel shows the amount of IgG light-chain (IgG-LC) in each IP. Lanes 1–2 : Input showing the amounts of LPA 1 and GIPC present in the lysates used for the IP. B, Upper panel : GIPC binds GST-LPA 1 (GST fused to the cytoplasmic tail of mouse LPA 1 (aa 311–364), lane 3) but not to GST alone (lane 2) or GST-LPA 1 ΔC (lacking the last three C-terminal amino acids, lane 4). Immobilized recombinant GST, GST-LPA 1 and GST- LPA 1 ΔC were incubated 4–15 h with lysates from HEK293 cells transiently transfected with FLAG-GIPC. Proteins bound to immobilized fusion proteins were eluted with 2X sample buffer for SDS-PAGE and immunoblotted with anti-GIPC IgG. Lane 1: input, showing the amount of GIPC in 1% of the lysate used for the assay. Lower panel : Ponceau staining demonstrating the amount of GST proteins used in each assay. C, Upper panel : Autoradiography showing that in vitro translated, [ 35 S]GIPC PDZ domain binds to GST-LPA 1 (lane 3) but not to GST alone (lane 2), GST- LPA 1 AAA (last three amino acids mutated to alanine, lane 4), or GST-LPA 2 (lane 5). GST fusion proteins were immobilized on glutathione-agarose beads as in “B” and incubated with in vitro translated [ 35 S]Met-labeled, GIPC PDZ domain (aa 125–225). Bound proteins were separated by SDS-PAGE and detected by autoradiography. Lane 1: 5% of the in vitro translated protein. Lower panel : Coomassie Blue staining showing the GST proteins used for the assay. D, Upper panel: Autoradiography showing that in vitro translated, [ 35 S] GIPC-PDZ interacts with the C-terminal PDZ binding motif of LPA 1 (SVV, lane 3) and with GST-GAIP (lane 8, used as a positive control [7] but shows little or no interaction with GST alone (lane 2) or GST-LPA 1 mutants in which the three C-terminal amino acids were modified to those of LPA 2 (STL, lane 4), LPA 3 (NGS, lane 5), LPA 4 (STF, lane 6) or LPA 5 (SAL, lane 7). Immobilized GST fusion proteins were incubated with in vitro translated [ 35 S]Met-labeled GIPC PDZ and analyzed as in C. Lower panel : Coomassie Blue staining showing the amounts of GST proteins used.

    Journal: PLoS ONE

    Article Title: The PDZ Protein GIPC Regulates Trafficking of the LPA1 Receptor from APPL Signaling Endosomes and Attenuates the Cell's Response to LPA

    doi: 10.1371/journal.pone.0049227

    Figure Lengend Snippet: GIPC directly interacts with the C-terminal PDZ binding motif of LPA 1 but not with other LPA receptors. A, Endogenous GIPC and GIPC-GFP co-immunoprecipitate with FLAG- LPA 1 from HEK cells expressing FLAG- LPA 1 (arrowhead, lane 4) but not control HEK cells (lane 3). C-terminally tagged GIPC-GFP and N-terminally tagged FLAG-LPA 1 were transiently coexpressed in HEK293 cells, and immunoprecipitation was carried out on cell lysates with mouse anti-FLAG IgG followed by immunoblotting with mouse anti-FLAG (LPA 1 ) and rabbit anti-GIPC IgG. Lanes were cropped from a single exposure of a continuous membrane. The lower panel shows the amount of IgG light-chain (IgG-LC) in each IP. Lanes 1–2 : Input showing the amounts of LPA 1 and GIPC present in the lysates used for the IP. B, Upper panel : GIPC binds GST-LPA 1 (GST fused to the cytoplasmic tail of mouse LPA 1 (aa 311–364), lane 3) but not to GST alone (lane 2) or GST-LPA 1 ΔC (lacking the last three C-terminal amino acids, lane 4). Immobilized recombinant GST, GST-LPA 1 and GST- LPA 1 ΔC were incubated 4–15 h with lysates from HEK293 cells transiently transfected with FLAG-GIPC. Proteins bound to immobilized fusion proteins were eluted with 2X sample buffer for SDS-PAGE and immunoblotted with anti-GIPC IgG. Lane 1: input, showing the amount of GIPC in 1% of the lysate used for the assay. Lower panel : Ponceau staining demonstrating the amount of GST proteins used in each assay. C, Upper panel : Autoradiography showing that in vitro translated, [ 35 S]GIPC PDZ domain binds to GST-LPA 1 (lane 3) but not to GST alone (lane 2), GST- LPA 1 AAA (last three amino acids mutated to alanine, lane 4), or GST-LPA 2 (lane 5). GST fusion proteins were immobilized on glutathione-agarose beads as in “B” and incubated with in vitro translated [ 35 S]Met-labeled, GIPC PDZ domain (aa 125–225). Bound proteins were separated by SDS-PAGE and detected by autoradiography. Lane 1: 5% of the in vitro translated protein. Lower panel : Coomassie Blue staining showing the GST proteins used for the assay. D, Upper panel: Autoradiography showing that in vitro translated, [ 35 S] GIPC-PDZ interacts with the C-terminal PDZ binding motif of LPA 1 (SVV, lane 3) and with GST-GAIP (lane 8, used as a positive control [7] but shows little or no interaction with GST alone (lane 2) or GST-LPA 1 mutants in which the three C-terminal amino acids were modified to those of LPA 2 (STL, lane 4), LPA 3 (NGS, lane 5), LPA 4 (STF, lane 6) or LPA 5 (SAL, lane 7). Immobilized GST fusion proteins were incubated with in vitro translated [ 35 S]Met-labeled GIPC PDZ and analyzed as in C. Lower panel : Coomassie Blue staining showing the amounts of GST proteins used.

    Article Snippet: Cell lysates (3–4 mg protein) were incubated at 4°C with mouse anti-FLAG IgG overnight followed by incubation with protein G-Sepharose beads (Sigma-Aldrich) for 1 h. Beads were then washed extensively with lysis buffer and resuspended in Laemmli sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue) for SDS-PAGE.

    Techniques: Binding Assay, Expressing, Immunoprecipitation, Recombinant, Incubation, Transfection, SDS Page, Staining, Autoradiography, In Vitro, Labeling, Positive Control, Modification, Next-Generation Sequencing

    GIPC depletion delays trafficking of LPA 1 from APPL1 to early EEA1 endosomes. A, GIPC depletion inhibits internalization of LPA 1 and its trafficking to early endosomes after stimulation with LPA. Upper panel: In HEK-LPA 1 cells transfected with control siRNA and stimulated with LPA for 15 min, LPA 1 is found in cytoplasmic vesicles where it colocalizes with EEA1 (arrowheads). Lower Panel : In cells transfected with GIPC siRNA fewer vesicles containing LPA 1 are present 15 min after LPA stimulation, and less colocalization is seen between LPA 1 and EEA1 (compare yellow in right panels). Boxed regions are enlarged (2.2×) in the insets. Images were acquired with a Zeiss AxioImager M1 microscope, and overlap in staining between LPA 1 and EEA1 was evaluated using Volocity software. Statistical significance (p value) was determined by t-test. B, Trafficking of LPA 1 is delayed in APPL1 endosomes after depletion of GIPC. Left panel: In both GIPC-depleted (GIPC siRNA) and controls (Ctrl siRNA), LPA 1 is localized along the plasma membrane after serum starvation (0 min) whereas APPL1 is found in peripheral cytoplasmic vesicles. Middle Panel : In both GIPC depleted and control cells stimulated with LPA for 3 min, LPA 1 colocalizes with APPL1 in cytoplasmic vesicles (arrowheads). Right Panel : In controls stimulated with LPA for 10 min, very few LPA 1 receptors remain in APPL endosomes (yellow, arrowhead) whereas in GIPC-depleted cells the majority of the receptors are retained in APPL endosomes (yellow, arrowhead). Boxed regions are enlarged (3×) in the insets. HEK-LPA 1 cells grown on coverslips were transfected with GIPC or control siRNA. 72 h after transfection cells were serum starved for 4–6 h and subsequently incubated on ice with rabbit (A) or mouse (C) anti-FLAG IgG, shifted to fresh medium containing LPA for the indicated times, then fixed and processed for immunofluorescence using mouse anti-EEA1 IgG (A) or rabbit-anti-APPL1 IgG (C) as in Fig. 2A. Images in “A” were acquired with a Zeiss AxioImage M1 microscope, and those in “C” were acquired with an Ultra View Vox Spinning Disk Confocal. Bar = 10 µm.

    Journal: PLoS ONE

    Article Title: The PDZ Protein GIPC Regulates Trafficking of the LPA1 Receptor from APPL Signaling Endosomes and Attenuates the Cell's Response to LPA

    doi: 10.1371/journal.pone.0049227

    Figure Lengend Snippet: GIPC depletion delays trafficking of LPA 1 from APPL1 to early EEA1 endosomes. A, GIPC depletion inhibits internalization of LPA 1 and its trafficking to early endosomes after stimulation with LPA. Upper panel: In HEK-LPA 1 cells transfected with control siRNA and stimulated with LPA for 15 min, LPA 1 is found in cytoplasmic vesicles where it colocalizes with EEA1 (arrowheads). Lower Panel : In cells transfected with GIPC siRNA fewer vesicles containing LPA 1 are present 15 min after LPA stimulation, and less colocalization is seen between LPA 1 and EEA1 (compare yellow in right panels). Boxed regions are enlarged (2.2×) in the insets. Images were acquired with a Zeiss AxioImager M1 microscope, and overlap in staining between LPA 1 and EEA1 was evaluated using Volocity software. Statistical significance (p value) was determined by t-test. B, Trafficking of LPA 1 is delayed in APPL1 endosomes after depletion of GIPC. Left panel: In both GIPC-depleted (GIPC siRNA) and controls (Ctrl siRNA), LPA 1 is localized along the plasma membrane after serum starvation (0 min) whereas APPL1 is found in peripheral cytoplasmic vesicles. Middle Panel : In both GIPC depleted and control cells stimulated with LPA for 3 min, LPA 1 colocalizes with APPL1 in cytoplasmic vesicles (arrowheads). Right Panel : In controls stimulated with LPA for 10 min, very few LPA 1 receptors remain in APPL endosomes (yellow, arrowhead) whereas in GIPC-depleted cells the majority of the receptors are retained in APPL endosomes (yellow, arrowhead). Boxed regions are enlarged (3×) in the insets. HEK-LPA 1 cells grown on coverslips were transfected with GIPC or control siRNA. 72 h after transfection cells were serum starved for 4–6 h and subsequently incubated on ice with rabbit (A) or mouse (C) anti-FLAG IgG, shifted to fresh medium containing LPA for the indicated times, then fixed and processed for immunofluorescence using mouse anti-EEA1 IgG (A) or rabbit-anti-APPL1 IgG (C) as in Fig. 2A. Images in “A” were acquired with a Zeiss AxioImage M1 microscope, and those in “C” were acquired with an Ultra View Vox Spinning Disk Confocal. Bar = 10 µm.

    Article Snippet: Cell lysates (3–4 mg protein) were incubated at 4°C with mouse anti-FLAG IgG overnight followed by incubation with protein G-Sepharose beads (Sigma-Aldrich) for 1 h. Beads were then washed extensively with lysis buffer and resuspended in Laemmli sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue) for SDS-PAGE.

    Techniques: Transfection, Microscopy, Staining, Software, Significance Assay, Incubation, Immunofluorescence

    Sandwich ELISA for detection of BoNT/B. MAb MCS6-27 was used as capture antibody, rabbit anti-BoNT/B polyclonal (Metabiologics) was used in conjunction with an HRP-conjugated, goat anti-rabbit polyclonal antibody as detector. Data are presented ±1 standard error, n = 3. Horizontal dashed lines represent the limit of detection (L.O.D., zero toxin plus 3 standard deviations) and the limit of quantitation (L.O.Q., zero toxin plus 5 standard deviations).

    Journal: PLoS ONE

    Article Title: Detection of Botulinum Neurotoxin Serotype B at Sub Mouse LD50 Levels by a Sandwich Immunoassay and Its Application to Toxin Detection in Milk

    doi: 10.1371/journal.pone.0011047

    Figure Lengend Snippet: Sandwich ELISA for detection of BoNT/B. MAb MCS6-27 was used as capture antibody, rabbit anti-BoNT/B polyclonal (Metabiologics) was used in conjunction with an HRP-conjugated, goat anti-rabbit polyclonal antibody as detector. Data are presented ±1 standard error, n = 3. Horizontal dashed lines represent the limit of detection (L.O.D., zero toxin plus 3 standard deviations) and the limit of quantitation (L.O.Q., zero toxin plus 5 standard deviations).

    Article Snippet: The plate was washed, then a 1 µg/mL solution of goat anti-mouse HRP-conjugated polyclonal antibodies #A4416 (Sigma-Aldrich) was added and the plates were incubated at 37°C for 1 h. Plates were again washed three times, then K-Blue substrate (Neogen Corporation, Lexington, KY) was added (100 µL per well) and incubated with agitation for 5 min at room temperature.

    Techniques: Sandwich ELISA, Quantitation Assay

    KLF9 interacts functionally and physically with the basic domain adjacent to the Cf of GATA4. (A) Schematic diagram of various GATA deletion mutants and the ability to stimulate the mouse Dio1 promoter in association with KLF9 and HNF4α as evaluated by luciferase reporter assay. TAD, transcriptional activation domain. The data shown are the mean of duplicate experiments. Error bars indicate the range of the duplicates. (B) Interaction of GATA4 and KLF9 in transfected HEK293 cells. HEK293 cells were transfected with expression plasmids for GATA4 along with FLAG-tagged KLF9. Cells were harvested, and whole-cell lysate were immunoprecipitated with polyclonal anti-GATA4 (AF2606; R D Systems) or control IgG (top) or monoclonal anti-FLAG M2 or control IgG (bottom) as described in Materials and Methods. The pellets from immunoprecipitation were subjected to SDS-PAGE and immunoblotted with anti-FLAG (top) or anti-GATA antibody (bottom). The expression of GATA4 or KLF9 was confirmed by immunoblot analysis of 10 μg of whole-cell lysate (Input) with anti-FLAG or anti-GATA4 antibody (AF2606), respectively. Ab, antibody; IP, immunoprecipitation; Cont, control. (C and D) Mapping of the region of GATA4 required for the interaction with KLF9 by coimmunoprecipitation analyses. HEK293 cells expressing FLAG-KLF9 and various GATA4 deletion mutants were immunoprecipitated with polyclonal antibody directed against the C-terminal part of GATA4 (aa 328 to 439) (sc-9053; Santa Cruz Biotechnology) (C) or the N-terminal part of GATA4 (aa 27 to 211) (AF2606) (D) as described for panel B. To detect KLF9 in the protein complex, peroxidase-conjugated anti-FLAG M2 antibody was used, and to detect GATA4, monoclonal anti-GATA4 (IgG-H2429) (for panel C) and polyclonal GATA4 (AF2606) antibodies (for panel D) were used for the immunoblot analyses. (E) Nuclear fractions were prepared from lysates of HEK293 cells expressing full-length (aa 1 to 442) or mutant (Δ304-332) GATA4. The presence of GATA4 in these subcellular fractions was detected by immunoblotting with an anti-GATA4 antibody. The nuclear fraction was confirmed by detection of the nuclear protein nucleoporin. (F) GST pull-down assays were performed with various 35 S-labeled GATA4 mutants in the presence of GST or GST fusions containing either the full length (aa 1 to 244) or the deletion mutant form of KLF9 [KLF9(Δ1-41)] immobilized on glutathione beads. After washing, specifically bound proteins were eluted, separated by SDS-PAGE, and detected by autoradiography. The input samples contain 1% of the material added to each GST assay. ZF, zinc finger.

    Journal: Molecular and Cellular Biology

    Article Title: Hepatocyte Nuclear Factor 4α Contributes to Thyroid Hormone Homeostasis by Cooperatively Regulating the Type 1 Iodothyronine Deiodinase Gene with GATA4 and Krüppel-Like Transcription Factor 9

    doi: 10.1128/MCB.02154-07

    Figure Lengend Snippet: KLF9 interacts functionally and physically with the basic domain adjacent to the Cf of GATA4. (A) Schematic diagram of various GATA deletion mutants and the ability to stimulate the mouse Dio1 promoter in association with KLF9 and HNF4α as evaluated by luciferase reporter assay. TAD, transcriptional activation domain. The data shown are the mean of duplicate experiments. Error bars indicate the range of the duplicates. (B) Interaction of GATA4 and KLF9 in transfected HEK293 cells. HEK293 cells were transfected with expression plasmids for GATA4 along with FLAG-tagged KLF9. Cells were harvested, and whole-cell lysate were immunoprecipitated with polyclonal anti-GATA4 (AF2606; R D Systems) or control IgG (top) or monoclonal anti-FLAG M2 or control IgG (bottom) as described in Materials and Methods. The pellets from immunoprecipitation were subjected to SDS-PAGE and immunoblotted with anti-FLAG (top) or anti-GATA antibody (bottom). The expression of GATA4 or KLF9 was confirmed by immunoblot analysis of 10 μg of whole-cell lysate (Input) with anti-FLAG or anti-GATA4 antibody (AF2606), respectively. Ab, antibody; IP, immunoprecipitation; Cont, control. (C and D) Mapping of the region of GATA4 required for the interaction with KLF9 by coimmunoprecipitation analyses. HEK293 cells expressing FLAG-KLF9 and various GATA4 deletion mutants were immunoprecipitated with polyclonal antibody directed against the C-terminal part of GATA4 (aa 328 to 439) (sc-9053; Santa Cruz Biotechnology) (C) or the N-terminal part of GATA4 (aa 27 to 211) (AF2606) (D) as described for panel B. To detect KLF9 in the protein complex, peroxidase-conjugated anti-FLAG M2 antibody was used, and to detect GATA4, monoclonal anti-GATA4 (IgG-H2429) (for panel C) and polyclonal GATA4 (AF2606) antibodies (for panel D) were used for the immunoblot analyses. (E) Nuclear fractions were prepared from lysates of HEK293 cells expressing full-length (aa 1 to 442) or mutant (Δ304-332) GATA4. The presence of GATA4 in these subcellular fractions was detected by immunoblotting with an anti-GATA4 antibody. The nuclear fraction was confirmed by detection of the nuclear protein nucleoporin. (F) GST pull-down assays were performed with various 35 S-labeled GATA4 mutants in the presence of GST or GST fusions containing either the full length (aa 1 to 244) or the deletion mutant form of KLF9 [KLF9(Δ1-41)] immobilized on glutathione beads. After washing, specifically bound proteins were eluted, separated by SDS-PAGE, and detected by autoradiography. The input samples contain 1% of the material added to each GST assay. ZF, zinc finger.

    Article Snippet: Monoclonal anti-FLAG M2 (F3165), peroxidase-conjugated anti-FLAG M2 (A8592), mouse monoclonal anti-β-actin (A1978), and affinity-purified goat anti-mouse IgG (A4416) were from Sigma.

    Techniques: Luciferase, Reporter Assay, Activation Assay, Transfection, Expressing, Immunoprecipitation, SDS Page, Mutagenesis, Labeling, Autoradiography, Glutathione S-Transferase Assay

    T 3 -regulated KLF9 activates the mouse Dio1 promoter. (A) Effects of T 3 treatment on various KLF mRNA levels in the normal mouse liver epithelial NMuLi cell line. On day 0, cells were set up at 5 × 10 5 /six-well plate in 2 ml of Dulbecco modified Eagle medium supplemented with 10% charcoal-dextran-treated fetal bovine serum (HyClone). On day 2, the cells were switched to the medium with or without 30 nM T 3. On day 3, cells were harvested for RNA analysis by QRT-PCR. (B) KLF9 activates the mouse Dio1 promoter. The indicated amounts of KLF9 expression plasmids were transfected into HepG2 cells together with pDio1(500) and incubated for 24 h, after which the cells were harvested and assayed for luciferase and β-galactosidase activities. The values above the closed circles refer to the level of induction relative to that found in the absence of the KLF9 expression plasmid. O.D., optical density. (C) Transactivation of the mouse Dio1 promoter by various KLF proteins in HepG2 cells. HepG2 cells were transfected with pDio1(500) and 0.1 μg of KLF expression plasmids. Luciferase assays were performed as described above. (D) Localization of KLF9-responsive element in the mouse Dio1 promoter by progressive deletion and mutation analyses. HepG2 cells were transfected with the indicated Dio1-luciferase reporter, in which each KLF motif was deleted or mutated, together with 0.1 μg of KLF9 expression plasmid, and assayed for transcriptional activity. The relative activation level (normalized luciferase activity cotransfected with KLF9 expression plasmid versus empty vector) is shown. (B to D) Each value represents the mean of duplicate experiments. Error bars indicate the range of the duplicates. (E) ChIP assays for KLF9 association with the Dio1 gene promoter. Cross-linked DNA-protein complexes from mouse liver extracts were immunoprecipitated with anit-KLF9 or control IgG, followed by PCR amplification with specific primers as schematically depicted in the diagram. Genomic DNA in the input cell lysates was used as a positive control.

    Journal: Molecular and Cellular Biology

    Article Title: Hepatocyte Nuclear Factor 4α Contributes to Thyroid Hormone Homeostasis by Cooperatively Regulating the Type 1 Iodothyronine Deiodinase Gene with GATA4 and Krüppel-Like Transcription Factor 9

    doi: 10.1128/MCB.02154-07

    Figure Lengend Snippet: T 3 -regulated KLF9 activates the mouse Dio1 promoter. (A) Effects of T 3 treatment on various KLF mRNA levels in the normal mouse liver epithelial NMuLi cell line. On day 0, cells were set up at 5 × 10 5 /six-well plate in 2 ml of Dulbecco modified Eagle medium supplemented with 10% charcoal-dextran-treated fetal bovine serum (HyClone). On day 2, the cells were switched to the medium with or without 30 nM T 3. On day 3, cells were harvested for RNA analysis by QRT-PCR. (B) KLF9 activates the mouse Dio1 promoter. The indicated amounts of KLF9 expression plasmids were transfected into HepG2 cells together with pDio1(500) and incubated for 24 h, after which the cells were harvested and assayed for luciferase and β-galactosidase activities. The values above the closed circles refer to the level of induction relative to that found in the absence of the KLF9 expression plasmid. O.D., optical density. (C) Transactivation of the mouse Dio1 promoter by various KLF proteins in HepG2 cells. HepG2 cells were transfected with pDio1(500) and 0.1 μg of KLF expression plasmids. Luciferase assays were performed as described above. (D) Localization of KLF9-responsive element in the mouse Dio1 promoter by progressive deletion and mutation analyses. HepG2 cells were transfected with the indicated Dio1-luciferase reporter, in which each KLF motif was deleted or mutated, together with 0.1 μg of KLF9 expression plasmid, and assayed for transcriptional activity. The relative activation level (normalized luciferase activity cotransfected with KLF9 expression plasmid versus empty vector) is shown. (B to D) Each value represents the mean of duplicate experiments. Error bars indicate the range of the duplicates. (E) ChIP assays for KLF9 association with the Dio1 gene promoter. Cross-linked DNA-protein complexes from mouse liver extracts were immunoprecipitated with anit-KLF9 or control IgG, followed by PCR amplification with specific primers as schematically depicted in the diagram. Genomic DNA in the input cell lysates was used as a positive control.

    Article Snippet: Monoclonal anti-FLAG M2 (F3165), peroxidase-conjugated anti-FLAG M2 (A8592), mouse monoclonal anti-β-actin (A1978), and affinity-purified goat anti-mouse IgG (A4416) were from Sigma.

    Techniques: Modification, Quantitative RT-PCR, Expressing, Transfection, Incubation, Luciferase, Plasmid Preparation, Mutagenesis, Activity Assay, Activation Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Positive Control

    The AF-2 domain of HNF4α and the Cf of GATA4 are required for both their association and the synergistic activation of the mouse Dio1 promoter with KLF9. (A) The Cf of GATA4 is required for its association with HNF4α, as demonstrated by mammalian two-hybrid assay. CHO cells were transfected with the reporter vector pGAL4-luc (0.15 μg) and hybrid constructs or empty plasmids (0.015 μg each) indicated. The values represent the mean luciferase activity of duplicate experiments. Error bars indicate the range of the duplicates. β-Galactosidase activity was used as an internal control. ZF, zinc finger; O.D., optical density. (B) Schematic diagram of the AF-2 deletion mutant [HNF4α(Δ359-368)] and the ability to bind to GATA4, as evaluated by coimmunoprecipitation analyses (bottom). Letters A to F in the diagram indicate conventional nuclear receptor domains. AF-2, activation function 2. Anti-HNF4α antibody (IgG-H1415) was used to detect HNF4α in protein complexes immunoprecipitated (IP) with an anti-GATA4 antibody (AF2606) from lysates of HEK293 cells expressing wild-type (wt) or AF-2 deletion mutant HNF4α along with GATA4. (C) Cotransfections were carried out with HepG2 cells with pDio1(500) and 0.05 μg of the wild type (wt) or the AF-2 deletion mutant (Δ359-368) of HNF4α together with either 0.05 μg of KLF9 or GATA4 expression plasmid or both together, as indicated. The values above the bars refer to the level of induction relative that found in the absence of the expression plasmid. The values represent the mean luciferase activity of duplicate experiments. Error bars indicate the range of the duplicates.

    Journal: Molecular and Cellular Biology

    Article Title: Hepatocyte Nuclear Factor 4α Contributes to Thyroid Hormone Homeostasis by Cooperatively Regulating the Type 1 Iodothyronine Deiodinase Gene with GATA4 and Krüppel-Like Transcription Factor 9

    doi: 10.1128/MCB.02154-07

    Figure Lengend Snippet: The AF-2 domain of HNF4α and the Cf of GATA4 are required for both their association and the synergistic activation of the mouse Dio1 promoter with KLF9. (A) The Cf of GATA4 is required for its association with HNF4α, as demonstrated by mammalian two-hybrid assay. CHO cells were transfected with the reporter vector pGAL4-luc (0.15 μg) and hybrid constructs or empty plasmids (0.015 μg each) indicated. The values represent the mean luciferase activity of duplicate experiments. Error bars indicate the range of the duplicates. β-Galactosidase activity was used as an internal control. ZF, zinc finger; O.D., optical density. (B) Schematic diagram of the AF-2 deletion mutant [HNF4α(Δ359-368)] and the ability to bind to GATA4, as evaluated by coimmunoprecipitation analyses (bottom). Letters A to F in the diagram indicate conventional nuclear receptor domains. AF-2, activation function 2. Anti-HNF4α antibody (IgG-H1415) was used to detect HNF4α in protein complexes immunoprecipitated (IP) with an anti-GATA4 antibody (AF2606) from lysates of HEK293 cells expressing wild-type (wt) or AF-2 deletion mutant HNF4α along with GATA4. (C) Cotransfections were carried out with HepG2 cells with pDio1(500) and 0.05 μg of the wild type (wt) or the AF-2 deletion mutant (Δ359-368) of HNF4α together with either 0.05 μg of KLF9 or GATA4 expression plasmid or both together, as indicated. The values above the bars refer to the level of induction relative that found in the absence of the expression plasmid. The values represent the mean luciferase activity of duplicate experiments. Error bars indicate the range of the duplicates.

    Article Snippet: Monoclonal anti-FLAG M2 (F3165), peroxidase-conjugated anti-FLAG M2 (A8592), mouse monoclonal anti-β-actin (A1978), and affinity-purified goat anti-mouse IgG (A4416) were from Sigma.

    Techniques: Activation Assay, Two Hybrid Assay, Transfection, Plasmid Preparation, Construct, Luciferase, Activity Assay, Mutagenesis, Immunoprecipitation, Expressing