br utp  (Millipore)


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  • 99
    Name:
    UTP
    Description:
    UTP is a 100 mM solution of the lithium salt pH 7 Contents UTP lithium salt 100 mM
    Catalog Number:
    11140949001
    Price:
    None
    Applications:
    UTP, lithium salt, is suitable for applications such as in vitro RNA transcription.
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    Structured Review

    Millipore br utp
    p68 mutation blocks gene shutoff during heat shock. ( A–D ) Control ( A,C ) and CB02119/Lip H ( B,D ) larvae were heat-shocked at 37°C for 20 min and immediately processed. The cells were stained using anti-H5 antibodies (green) and anti-HSF antibodies (red) (cf. C and D ). Examining the anti-H5 staining alone reveals that active Pol II is limited to major heat-shock puffs in control cells ( C ), while in p68 mutant cells, it is present at heat-shock loci, other discrete sites, and generally along chromosome arms ( D ) ( hsp70 gene loci at 87A and 87C are indicated). ( E–F ) Control ( E,G ) and CB02119/Lip H ( F,H ) larvae were heat-shocked at 37°C for 10 min, pulse-labeled with <t>Br-UTP,</t> and heat-shocked for an additional 20 min. ( E,G ) Control samples exhibit Br-UTP labeling only at heat-shock response puffs. ( F,H ) p68 mutant cells show high levels of Br-UTP incorporation at many chromosomal sites including the nucleolus, demonstrating that a broad number of genes continue to transcribe <t>RNA</t> during heat shock in the absence of p68.
    UTP is a 100 mM solution of the lithium salt pH 7 Contents UTP lithium salt 100 mM
    https://www.bioz.com/result/br utp/product/Millipore
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    br utp - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "The Drosophila P68 RNA helicase regulates transcriptional deactivation by promoting RNA release from chromatin"

    Article Title: The Drosophila P68 RNA helicase regulates transcriptional deactivation by promoting RNA release from chromatin

    Journal: Genes & Development

    doi: 10.1101/gad.1396306

    p68 mutation blocks gene shutoff during heat shock. ( A–D ) Control ( A,C ) and CB02119/Lip H ( B,D ) larvae were heat-shocked at 37°C for 20 min and immediately processed. The cells were stained using anti-H5 antibodies (green) and anti-HSF antibodies (red) (cf. C and D ). Examining the anti-H5 staining alone reveals that active Pol II is limited to major heat-shock puffs in control cells ( C ), while in p68 mutant cells, it is present at heat-shock loci, other discrete sites, and generally along chromosome arms ( D ) ( hsp70 gene loci at 87A and 87C are indicated). ( E–F ) Control ( E,G ) and CB02119/Lip H ( F,H ) larvae were heat-shocked at 37°C for 10 min, pulse-labeled with Br-UTP, and heat-shocked for an additional 20 min. ( E,G ) Control samples exhibit Br-UTP labeling only at heat-shock response puffs. ( F,H ) p68 mutant cells show high levels of Br-UTP incorporation at many chromosomal sites including the nucleolus, demonstrating that a broad number of genes continue to transcribe RNA during heat shock in the absence of p68.
    Figure Legend Snippet: p68 mutation blocks gene shutoff during heat shock. ( A–D ) Control ( A,C ) and CB02119/Lip H ( B,D ) larvae were heat-shocked at 37°C for 20 min and immediately processed. The cells were stained using anti-H5 antibodies (green) and anti-HSF antibodies (red) (cf. C and D ). Examining the anti-H5 staining alone reveals that active Pol II is limited to major heat-shock puffs in control cells ( C ), while in p68 mutant cells, it is present at heat-shock loci, other discrete sites, and generally along chromosome arms ( D ) ( hsp70 gene loci at 87A and 87C are indicated). ( E–F ) Control ( E,G ) and CB02119/Lip H ( F,H ) larvae were heat-shocked at 37°C for 10 min, pulse-labeled with Br-UTP, and heat-shocked for an additional 20 min. ( E,G ) Control samples exhibit Br-UTP labeling only at heat-shock response puffs. ( F,H ) p68 mutant cells show high levels of Br-UTP incorporation at many chromosomal sites including the nucleolus, demonstrating that a broad number of genes continue to transcribe RNA during heat shock in the absence of p68.

    Techniques Used: Mutagenesis, Staining, Labeling

    p68 mutants display RNA export defects. ( A,B ) Control ( A ) and CB02119/Lip F ( B ) transheterozygotes labeled for hsp70 RNA immediately after a 20-min heat shock. The edge of representative nuclei is outlined in white (boxes). p68 mutant salivary glands have less cytoplasmic hsp70 RNA and qualitatively higher levels of hsp70 RNA at transcription sites. ( C,D ) Control ( C ) and CB02119/Lip F ( D ) salivary glands labeled with Br-UTP. Control cells exhibit cytoplasmic staining and punctuate nuclear Br-UTP staining, whereas p68 mutant salivary glands display robust Br-UTP labeling on polytene chromosomal bands. Little signal was observed in the cytoplasm of mutant cells. ( E–H ) Control ( E,G ) and CB02119/Lip F ( F,H ) salivary gland cells stained with anti-SBR (NXF1) antibodies ( E,F ) or anti-ALY (REF1) antibodies ( G,H ). SBR and ALY are found along the nuclear periphery and in the nucleoplasm of control cells. In p68 mutant cells, their distribution is changed, and both export factors accumulate in the nuclear interior.
    Figure Legend Snippet: p68 mutants display RNA export defects. ( A,B ) Control ( A ) and CB02119/Lip F ( B ) transheterozygotes labeled for hsp70 RNA immediately after a 20-min heat shock. The edge of representative nuclei is outlined in white (boxes). p68 mutant salivary glands have less cytoplasmic hsp70 RNA and qualitatively higher levels of hsp70 RNA at transcription sites. ( C,D ) Control ( C ) and CB02119/Lip F ( D ) salivary glands labeled with Br-UTP. Control cells exhibit cytoplasmic staining and punctuate nuclear Br-UTP staining, whereas p68 mutant salivary glands display robust Br-UTP labeling on polytene chromosomal bands. Little signal was observed in the cytoplasm of mutant cells. ( E–H ) Control ( E,G ) and CB02119/Lip F ( F,H ) salivary gland cells stained with anti-SBR (NXF1) antibodies ( E,F ) or anti-ALY (REF1) antibodies ( G,H ). SBR and ALY are found along the nuclear periphery and in the nucleoplasm of control cells. In p68 mutant cells, their distribution is changed, and both export factors accumulate in the nuclear interior.

    Techniques Used: Labeling, Mutagenesis, Staining

    2) Product Images from "Nuclear ErbB-2 Enhances Translation and Cell Growth by activating transcription of rRNA genes"

    Article Title: Nuclear ErbB-2 Enhances Translation and Cell Growth by activating transcription of rRNA genes

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-10-3504

    Nuclear ErbB2 increases RNA Pol I transcription in vivo. A, Cells transfected with ErbB2 or non-specific (NS) siRNAs were assessed for 45S pre-rRNA synthesis by RT-qPCR (top) and ErbB2 protein expression (bottom). error bar, SD; B, SKBR3 and HER18 cells transfected with ErbB2 siRNA or NS siRNA were subjected to Br-UTP incorporation assays of nascent nucleolar RNA and confocal microscopy for ErbB-2 (green), Br-UTP (red) and nuclei (DAPI, blue). Representative images are from SKBR3 cells. Percent of Br-UTP positive cells shown as means with SD. C, Permeabilized SKBR3 cells were incubated with Br-UTP to label active transcription sites with or without α-amanitin. Confocal microscopy was as in B. D, left, Cells were examined for 45S pre-rRNA synthesis (top) and ErbB2 protein (bottom) as in A. Relative amounts of 45S pre-rRNA shown as means with SD. Middle, Br-UTP incorporation assays of nascent nucleolar RNA in MCF-7 and the MCF-7 stable cell line expressing wild-type ErbB2 (HER18). Percent of Br-UTP positive cells shown as means with SD. Right, Cells transfected with increasing amounts of ErbB2 were measured for 45S pre-rRNA synthesis (top) and ErbB2 protein (middle) as in A, and RT-PCR of pre-rRNA and internal control GAPDH (bottom). E, Transient (293) or stable (MCF-7 and MDA-MB 231) transfectants of wild-type ErbB-2 (WT), ErbB-2Δ NLS mutant or vector (Vec) were assayed for co-IP of ErbB-2 with RNA Pol I (left), 45S pre-rRNA synthesis and ErbB2 protein (right) as in D (right).
    Figure Legend Snippet: Nuclear ErbB2 increases RNA Pol I transcription in vivo. A, Cells transfected with ErbB2 or non-specific (NS) siRNAs were assessed for 45S pre-rRNA synthesis by RT-qPCR (top) and ErbB2 protein expression (bottom). error bar, SD; B, SKBR3 and HER18 cells transfected with ErbB2 siRNA or NS siRNA were subjected to Br-UTP incorporation assays of nascent nucleolar RNA and confocal microscopy for ErbB-2 (green), Br-UTP (red) and nuclei (DAPI, blue). Representative images are from SKBR3 cells. Percent of Br-UTP positive cells shown as means with SD. C, Permeabilized SKBR3 cells were incubated with Br-UTP to label active transcription sites with or without α-amanitin. Confocal microscopy was as in B. D, left, Cells were examined for 45S pre-rRNA synthesis (top) and ErbB2 protein (bottom) as in A. Relative amounts of 45S pre-rRNA shown as means with SD. Middle, Br-UTP incorporation assays of nascent nucleolar RNA in MCF-7 and the MCF-7 stable cell line expressing wild-type ErbB2 (HER18). Percent of Br-UTP positive cells shown as means with SD. Right, Cells transfected with increasing amounts of ErbB2 were measured for 45S pre-rRNA synthesis (top) and ErbB2 protein (middle) as in A, and RT-PCR of pre-rRNA and internal control GAPDH (bottom). E, Transient (293) or stable (MCF-7 and MDA-MB 231) transfectants of wild-type ErbB-2 (WT), ErbB-2Δ NLS mutant or vector (Vec) were assayed for co-IP of ErbB-2 with RNA Pol I (left), 45S pre-rRNA synthesis and ErbB2 protein (right) as in D (right).

    Techniques Used: In Vivo, Transfection, Quantitative RT-PCR, Expressing, Confocal Microscopy, Incubation, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Multiple Displacement Amplification, Mutagenesis, Plasmid Preparation, Co-Immunoprecipitation Assay

    Related Articles

    Modification:

    Article Title: Pseudomonas aeruginosa Quorum-Sensing Signal Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone Inhibits Expression of P2Y Receptors in Cystic Fibrosis Tracheal Gland Cells
    Article Snippet: .. ATP, UTP, epinephrine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), indomethacin, nordihydrogaiaretic acid (NDGA), dexamethasone, cortisone, prednisone, ibuprofen, and Dulbecco’s modified Eagle’s medium-Ham’s F12 medium were obtained from Sigma (St. Louis, Mo.). ..

    MTT Assay:

    Article Title: Pseudomonas aeruginosa Quorum-Sensing Signal Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone Inhibits Expression of P2Y Receptors in Cystic Fibrosis Tracheal Gland Cells
    Article Snippet: .. ATP, UTP, epinephrine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), indomethacin, nordihydrogaiaretic acid (NDGA), dexamethasone, cortisone, prednisone, ibuprofen, and Dulbecco’s modified Eagle’s medium-Ham’s F12 medium were obtained from Sigma (St. Louis, Mo.). ..

    other:

    Article Title: Calcium Transduces Plasma Membrane Receptor Signals to Produce Diacylglycerol at Golgi Membranes *
    Article Snippet: Phorbol 12,13-dibutyrate (PDBu), edelfosine, thapsigargin, propranolol, and UTP were obtained from Calbiochem.

    Article Title: UTP – Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells
    Article Snippet: ATP, UTP, UDP were from Sigma–Aldrich (St. Louis, MO, United States) and UTPγS, thapsigargin, U73122, U73343, GF109203X, α,β-MeATP, MRS compounds and XE-991 dihydrochloride were from TOCRIS Bioscience (Bristol, United Kingdom).

    Article Title: UTP is not a biased agonist at human P2Y11 receptors
    Article Snippet: ATP (disodium salt) and UTP (sodium salt) (Sigma/RBI, UK) were dissolved in deionised water as 10 mM stock solutions and diluted in buffer before use.

    Article Title: Inhibition of Apoptosis by P2Y2 Receptor Activation: Novel Pathways for Neuronal Survival
    Article Snippet: The following reagents were used: ATPγS, ATP, UTP, histamine (Sigma, St. Louis, MO), NGF (Invitrogen, Carlsbad, CA), methyl-9-( S )-12( R )-epoxy-1 H -diindolo[1,2,3-fg: 3′2′1′-kl]pyrrolo[3,4- i ][1,6]benzodiazocine-2,3,9,10,11,12-hexahydro-10-( R )-hydroxy-9-methyl-1-oxo-10-carboxilate (K252a), 4-amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo[3,4- d ]pyrimidine (PP2), 3-[1–3-(amidinothio)-propyl-1 H -indol-3-yl]-3-(1-methyl-1 H -indol-3-yl)maleimide (Ro-31-8220), 2-(2-diamino-3-methoxyphenyl-4 H -1-benzopyran-4-one (PD98059), 2,3-dihydro- N , N -dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1 H -indol-2-yl)methylene]-1 H -indole-5-sulfonamide (SU6656), 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1 H -pyrrole-2,5-dione , BAPTA-AM, Raf kinase inhibitor (Calbiochem, San Diego, CA), 1,4-diamino-2,3-dicyano-1,4-bis( o -aminophenylmercapto)butadiene (U0126), and 2-(4-morpholinyl)-8-phenyl-1(4 H )-benzopyran-4-one ( ) (Cell Signaling, Beverly, MA).

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  • 99
    Millipore utp
    A kinetic model simulates exocytosis. (A) Cartoon of the vesicle pools and transitions assumed in the model. (B) Formal kinetic diagram corresponding to cartoon in A and showing the stimulus conditions that affect each step. (C) Comparison of simulated time courses of the rate of exocytosis (blue lines) with recorded data from previous figures (filled circles). The left column is without and the right column with FSK treatment under five different stimuli. (C, a and b) Exocytosis evoked by <t>ionomycin-mediated</t> Ca 2+ (same data as in Fig. 2 B ). (C, c–h) Exocytosis induced by different concentrations of <t>UTP</t> (from Fig. 4 ). (C, i–j) Exocytosis triggered by 100 nM trypsin (from Fig. 3 D ).
    Utp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/utp/product/Millipore
    Average 99 stars, based on 92 article reviews
    Price from $9.99 to $1999.99
    utp - by Bioz Stars, 2020-09
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    97
    Millipore ionomycin
    Granules migrate in response to [Ca 2+ ] i rise. Before each experiment, cells were preincubated with 1 μM <t>ionomycin</t> for at least 5 min in Ca 2+ -free solution (0 Ca 2+ ), and all test solutions contained 1 μM ionomycin in the Ca 2+ -free solution. (A) TIRFM images obtained just before (a) and during (b) 2 mM Ca 2+ (2 Ca 2+ ). Bars, 4 μm. (B) Trajectory in the x–y plane of the two granules indicated in A before (black) and during (red) 2 mM Ca 2+ treatment. Scale bars are in units of micrometers. (C) Cumulative distance traveled in the x–y plane. (D) Speed in the x–y plane. (E) Change of fluorescence intensity of two representative granules in an arbitrary unit (A.U.). Increase of fluorescence intensity represents migration of the granules to the plasma membrane. (F) Intensity of 26 randomly chosen granules from A. All data are sampled at 2-s intervals. For fluorescence intensity of individual granules in E and F, background fluorescence from a granule-free region was subtracted.
    Ionomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 5718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ionomycin/product/Millipore
    Average 97 stars, based on 5718 article reviews
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    93
    Millipore anti erbb 2
    Nuclear ErbB2 increases protein synthesis and cell size. A, left, Total protein contents from equal cell numbers of MCF-7 and HER18 cells were determined by the Bradford method. Right, 35 S-methionine labeling of protein synthesis in equal cell numbers of MCF-7 and HER18 cells were measured by scintillation counting. B, Knockdown of ErbB2 using siRNA results in decreased protein biosynthesis. Cells transfected with ErbB2 or non-specific (NS) siRNAs were monitored for total protein concentration (left, top) and 35 S-methionine incorporation (right, top) as analyzed in A. ErbB2 protein knockdown was confirmed by western blotting (bottom). C and D, Total protein level (C) and cell size (D) are increased in wild-type (WT) <t>ErbB-2-expressing</t> cells but not in cells expressing ErbB-2Δ NLS mutant. MCF-7 and MDA-MB-231 cells expressing WT ErbB-2, ErbB-2Δ NLS mutant or vector control were assayed for total protein synthesis (C, top) and ErbB2 protein expression (C, bottom) as in B. Cells were also analyzed by flow cytometry to detect DNA content and cell size of G1-, S- and G2/M- phase cells using the parameter mean forward scatter height (FSC-H), which is a measure of relative cell size (D). P value was analyzed by Student’s t test.
    Anti Erbb 2, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A kinetic model simulates exocytosis. (A) Cartoon of the vesicle pools and transitions assumed in the model. (B) Formal kinetic diagram corresponding to cartoon in A and showing the stimulus conditions that affect each step. (C) Comparison of simulated time courses of the rate of exocytosis (blue lines) with recorded data from previous figures (filled circles). The left column is without and the right column with FSK treatment under five different stimuli. (C, a and b) Exocytosis evoked by ionomycin-mediated Ca 2+ (same data as in Fig. 2 B ). (C, c–h) Exocytosis induced by different concentrations of UTP (from Fig. 4 ). (C, i–j) Exocytosis triggered by 100 nM trypsin (from Fig. 3 D ).

    Journal: The Journal of General Physiology

    Article Title: Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

    doi: 10.1085/jgp.200910355

    Figure Lengend Snippet: A kinetic model simulates exocytosis. (A) Cartoon of the vesicle pools and transitions assumed in the model. (B) Formal kinetic diagram corresponding to cartoon in A and showing the stimulus conditions that affect each step. (C) Comparison of simulated time courses of the rate of exocytosis (blue lines) with recorded data from previous figures (filled circles). The left column is without and the right column with FSK treatment under five different stimuli. (C, a and b) Exocytosis evoked by ionomycin-mediated Ca 2+ (same data as in Fig. 2 B ). (C, c–h) Exocytosis induced by different concentrations of UTP (from Fig. 4 ). (C, i–j) Exocytosis triggered by 100 nM trypsin (from Fig. 3 D ).

    Article Snippet: UTP, ionomycin, H-89, and Rp-8-Br-cAMPS were obtained from Calbiochem.

    Techniques:

    FSK evokes cAMP increase. Optical measurements of cAMP production in PDECs transfected with Epac1-camps, a FRET probe. (A) Time courses of YFP (dotted olive line) and CFP (dotted cyan line) fluorescence from a single cell treated with 20 μM FSK to stimulate adenylyl cyclase. When the FRET ratio (F YFP /F CFP ; red line, plotted on a reversed axis) decreases, cytoplasmic cAMP concentration increases. (B) Mean normalized (Norm.) FRET ratio with 20 μM FSK (red line, n = 6) or 100 μM UTP (black line, n = 9). The gray bar indicates the duration of treatment with FSK or UTP in normal control solution. (C) The effect of 1 μM FSK on cAMP production in cells exposed to a solution free of Ca 2+ (0 Ca 2+ , including 100 μM EGTA) in the presence 1 μM ionomycin for at least 5 min, and then treated with solution containing 2 mM Ca 2+ (2 Ca 2+ , black bar). In this measurement, we used 1 μM FSK as in the later experiments. n = 5.

    Journal: The Journal of General Physiology

    Article Title: Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

    doi: 10.1085/jgp.200910355

    Figure Lengend Snippet: FSK evokes cAMP increase. Optical measurements of cAMP production in PDECs transfected with Epac1-camps, a FRET probe. (A) Time courses of YFP (dotted olive line) and CFP (dotted cyan line) fluorescence from a single cell treated with 20 μM FSK to stimulate adenylyl cyclase. When the FRET ratio (F YFP /F CFP ; red line, plotted on a reversed axis) decreases, cytoplasmic cAMP concentration increases. (B) Mean normalized (Norm.) FRET ratio with 20 μM FSK (red line, n = 6) or 100 μM UTP (black line, n = 9). The gray bar indicates the duration of treatment with FSK or UTP in normal control solution. (C) The effect of 1 μM FSK on cAMP production in cells exposed to a solution free of Ca 2+ (0 Ca 2+ , including 100 μM EGTA) in the presence 1 μM ionomycin for at least 5 min, and then treated with solution containing 2 mM Ca 2+ (2 Ca 2+ , black bar). In this measurement, we used 1 μM FSK as in the later experiments. n = 5.

    Article Snippet: UTP, ionomycin, H-89, and Rp-8-Br-cAMPS were obtained from Calbiochem.

    Techniques: Transfection, Fluorescence, Concentration Assay

    Granules migrate in response to [Ca 2+ ] i rise. Before each experiment, cells were preincubated with 1 μM ionomycin for at least 5 min in Ca 2+ -free solution (0 Ca 2+ ), and all test solutions contained 1 μM ionomycin in the Ca 2+ -free solution. (A) TIRFM images obtained just before (a) and during (b) 2 mM Ca 2+ (2 Ca 2+ ). Bars, 4 μm. (B) Trajectory in the x–y plane of the two granules indicated in A before (black) and during (red) 2 mM Ca 2+ treatment. Scale bars are in units of micrometers. (C) Cumulative distance traveled in the x–y plane. (D) Speed in the x–y plane. (E) Change of fluorescence intensity of two representative granules in an arbitrary unit (A.U.). Increase of fluorescence intensity represents migration of the granules to the plasma membrane. (F) Intensity of 26 randomly chosen granules from A. All data are sampled at 2-s intervals. For fluorescence intensity of individual granules in E and F, background fluorescence from a granule-free region was subtracted.

    Journal: The Journal of General Physiology

    Article Title: Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

    doi: 10.1085/jgp.200910355

    Figure Lengend Snippet: Granules migrate in response to [Ca 2+ ] i rise. Before each experiment, cells were preincubated with 1 μM ionomycin for at least 5 min in Ca 2+ -free solution (0 Ca 2+ ), and all test solutions contained 1 μM ionomycin in the Ca 2+ -free solution. (A) TIRFM images obtained just before (a) and during (b) 2 mM Ca 2+ (2 Ca 2+ ). Bars, 4 μm. (B) Trajectory in the x–y plane of the two granules indicated in A before (black) and during (red) 2 mM Ca 2+ treatment. Scale bars are in units of micrometers. (C) Cumulative distance traveled in the x–y plane. (D) Speed in the x–y plane. (E) Change of fluorescence intensity of two representative granules in an arbitrary unit (A.U.). Increase of fluorescence intensity represents migration of the granules to the plasma membrane. (F) Intensity of 26 randomly chosen granules from A. All data are sampled at 2-s intervals. For fluorescence intensity of individual granules in E and F, background fluorescence from a granule-free region was subtracted.

    Article Snippet: UTP, ionomycin, H-89, and Rp-8-Br-cAMPS were obtained from Calbiochem.

    Techniques: Fluorescence, Migration

    cAMP does not affect granule migration. Before each experiment, cells were preincubated with 1 μM ionomycin for at least 5 min in Ca 2+ -free solution (0 Ca 2+ ), and all test solutions contained 1 μM ionomycin in a Ca 2+ -free solution. Treatments with different concentrations of extracellular Ca 2+ and with FSK are indicated by bars at the top of each graph. The open, black, and gray bars indicate 0 mM Ca 2+ (0 Ca 2+ ), 2 mM Ca 2+ (2 Ca 2+ ), and 1 μM FSK, respectively. (A and C) Mean time course of intensity of granules (A , N = 24, and C , N = 10, black line) approaching to the plasma membrane during an increase in [Ca 2+ ] i (A, n = 13, and C , n = 11, green line) in the absence (A) or presence (C) of 1 μM FSK. (B) Mean time course of intensity change of granules (black line) and [Ca 2+ ] i (green line) after the FSK treatment ( n = 11). (D) Mean time course of return of granule intensity after Ca 2+ removal. The black and red lines indicate kinetics of granule moving away from the plasma membrane in the absence or presence of FSK, respectively. [Ca 2+ ] i level is shown as green dotted line without FSK and green solid line with FSK. (E) Number of granules near the plasma membrane under the conditions 0 Ca 2+ , 2 Ca 2+ ( n = 23), FSK + 0 Ca 2+ ( n = 9), and FSK + 2 Ca 2+ ( n = 9), normalized to that in 0 Ca 2+ in each experiment.

    Journal: The Journal of General Physiology

    Article Title: Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

    doi: 10.1085/jgp.200910355

    Figure Lengend Snippet: cAMP does not affect granule migration. Before each experiment, cells were preincubated with 1 μM ionomycin for at least 5 min in Ca 2+ -free solution (0 Ca 2+ ), and all test solutions contained 1 μM ionomycin in a Ca 2+ -free solution. Treatments with different concentrations of extracellular Ca 2+ and with FSK are indicated by bars at the top of each graph. The open, black, and gray bars indicate 0 mM Ca 2+ (0 Ca 2+ ), 2 mM Ca 2+ (2 Ca 2+ ), and 1 μM FSK, respectively. (A and C) Mean time course of intensity of granules (A , N = 24, and C , N = 10, black line) approaching to the plasma membrane during an increase in [Ca 2+ ] i (A, n = 13, and C , n = 11, green line) in the absence (A) or presence (C) of 1 μM FSK. (B) Mean time course of intensity change of granules (black line) and [Ca 2+ ] i (green line) after the FSK treatment ( n = 11). (D) Mean time course of return of granule intensity after Ca 2+ removal. The black and red lines indicate kinetics of granule moving away from the plasma membrane in the absence or presence of FSK, respectively. [Ca 2+ ] i level is shown as green dotted line without FSK and green solid line with FSK. (E) Number of granules near the plasma membrane under the conditions 0 Ca 2+ , 2 Ca 2+ ( n = 23), FSK + 0 Ca 2+ ( n = 9), and FSK + 2 Ca 2+ ( n = 9), normalized to that in 0 Ca 2+ in each experiment.

    Article Snippet: UTP, ionomycin, H-89, and Rp-8-Br-cAMPS were obtained from Calbiochem.

    Techniques: Migration

    FSK potentiates Ca 2+ -evoked exocytosis. ( A and C ) Cells, loaded sequentially with dopamine for amperometry and then with indo-1 Ca 2+ -sensitive dye, were pretreated with 1 μM ionomycin in Ca 2+ -free solution (0 Ca 2+ ). All test solutions contained 1 μM ionomycin in a Ca 2+ -free solution. Increases of [Ca 2+ ] i and of exocytosis were induced by switching to 2 mM Ca 2+ (2 Ca 2+ ). Top traces are representative amperometric current recordings. The lower traces show optically measured [Ca 2+ ] i (green line) and the simultaneous rate of exocytosis (filled circles, number of spikes per 30 s) in the absence (A) or presence (C) of FSK. (B and D) Average time course of [Ca 2+ ] i (green line) and normalized rate of exocytosis (symbols) in the absence (B; n = 6) or presence (D; n = 12) of 1 μM FSK.

    Journal: The Journal of General Physiology

    Article Title: Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

    doi: 10.1085/jgp.200910355

    Figure Lengend Snippet: FSK potentiates Ca 2+ -evoked exocytosis. ( A and C ) Cells, loaded sequentially with dopamine for amperometry and then with indo-1 Ca 2+ -sensitive dye, were pretreated with 1 μM ionomycin in Ca 2+ -free solution (0 Ca 2+ ). All test solutions contained 1 μM ionomycin in a Ca 2+ -free solution. Increases of [Ca 2+ ] i and of exocytosis were induced by switching to 2 mM Ca 2+ (2 Ca 2+ ). Top traces are representative amperometric current recordings. The lower traces show optically measured [Ca 2+ ] i (green line) and the simultaneous rate of exocytosis (filled circles, number of spikes per 30 s) in the absence (A) or presence (C) of FSK. (B and D) Average time course of [Ca 2+ ] i (green line) and normalized rate of exocytosis (symbols) in the absence (B; n = 6) or presence (D; n = 12) of 1 μM FSK.

    Article Snippet: UTP, ionomycin, H-89, and Rp-8-Br-cAMPS were obtained from Calbiochem.

    Techniques:

    A kinetic model simulates exocytosis. (A) Cartoon of the vesicle pools and transitions assumed in the model. (B) Formal kinetic diagram corresponding to cartoon in A and showing the stimulus conditions that affect each step. (C) Comparison of simulated time courses of the rate of exocytosis (blue lines) with recorded data from previous figures (filled circles). The left column is without and the right column with FSK treatment under five different stimuli. (C, a and b) Exocytosis evoked by ionomycin-mediated Ca 2+ (same data as in Fig. 2 B ). (C, c–h) Exocytosis induced by different concentrations of UTP (from Fig. 4 ). (C, i–j) Exocytosis triggered by 100 nM trypsin (from Fig. 3 D ).

    Journal: The Journal of General Physiology

    Article Title: Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

    doi: 10.1085/jgp.200910355

    Figure Lengend Snippet: A kinetic model simulates exocytosis. (A) Cartoon of the vesicle pools and transitions assumed in the model. (B) Formal kinetic diagram corresponding to cartoon in A and showing the stimulus conditions that affect each step. (C) Comparison of simulated time courses of the rate of exocytosis (blue lines) with recorded data from previous figures (filled circles). The left column is without and the right column with FSK treatment under five different stimuli. (C, a and b) Exocytosis evoked by ionomycin-mediated Ca 2+ (same data as in Fig. 2 B ). (C, c–h) Exocytosis induced by different concentrations of UTP (from Fig. 4 ). (C, i–j) Exocytosis triggered by 100 nM trypsin (from Fig. 3 D ).

    Article Snippet: UTP, ionomycin, H-89, and Rp-8-Br-cAMPS were obtained from Calbiochem.

    Techniques:

    FSK evokes cAMP increase. Optical measurements of cAMP production in PDECs transfected with Epac1-camps, a FRET probe. (A) Time courses of YFP (dotted olive line) and CFP (dotted cyan line) fluorescence from a single cell treated with 20 μM FSK to stimulate adenylyl cyclase. When the FRET ratio (F YFP /F CFP ; red line, plotted on a reversed axis) decreases, cytoplasmic cAMP concentration increases. (B) Mean normalized (Norm.) FRET ratio with 20 μM FSK (red line, n = 6) or 100 μM UTP (black line, n = 9). The gray bar indicates the duration of treatment with FSK or UTP in normal control solution. (C) The effect of 1 μM FSK on cAMP production in cells exposed to a solution free of Ca 2+ (0 Ca 2+ , including 100 μM EGTA) in the presence 1 μM ionomycin for at least 5 min, and then treated with solution containing 2 mM Ca 2+ (2 Ca 2+ , black bar). In this measurement, we used 1 μM FSK as in the later experiments. n = 5.

    Journal: The Journal of General Physiology

    Article Title: Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

    doi: 10.1085/jgp.200910355

    Figure Lengend Snippet: FSK evokes cAMP increase. Optical measurements of cAMP production in PDECs transfected with Epac1-camps, a FRET probe. (A) Time courses of YFP (dotted olive line) and CFP (dotted cyan line) fluorescence from a single cell treated with 20 μM FSK to stimulate adenylyl cyclase. When the FRET ratio (F YFP /F CFP ; red line, plotted on a reversed axis) decreases, cytoplasmic cAMP concentration increases. (B) Mean normalized (Norm.) FRET ratio with 20 μM FSK (red line, n = 6) or 100 μM UTP (black line, n = 9). The gray bar indicates the duration of treatment with FSK or UTP in normal control solution. (C) The effect of 1 μM FSK on cAMP production in cells exposed to a solution free of Ca 2+ (0 Ca 2+ , including 100 μM EGTA) in the presence 1 μM ionomycin for at least 5 min, and then treated with solution containing 2 mM Ca 2+ (2 Ca 2+ , black bar). In this measurement, we used 1 μM FSK as in the later experiments. n = 5.

    Article Snippet: UTP, ionomycin, H-89, and Rp-8-Br-cAMPS were obtained from Calbiochem.

    Techniques: Transfection, Fluorescence, Concentration Assay

    cAMP does not affect granule mobility. Before each experiment, cells were preincubated with 1 μM ionomycin for at least 5 min in Ca 2+ -free solution (0 Ca 2+ ), and all test solutions contained 1 μM ionomycin in a Ca 2+ -free solution. Average speed of granule movement during treatment with (A) 2 mM Ca 2+ (‘2 Ca 2+ ’, n = 2, n = 19), (B) 1 μM FSK ( n = 3, n = 30), and (C) 2 mM Ca 2+ with FSK ( n = 3, n = 25), where N and n indicate the number of cells and granules for each experiment. External Ca 2+ concentration was exchanged from 0 to 2 mM Ca 2+ to increase [Ca 2+ ] i . Red dotted lines indicate average granule speed in control conditions before external Ca 2+ or FSK treatments. Average MSD of the same granules is plotted on the right side of each figure. The same color coding for the treatments is used.

    Journal: The Journal of General Physiology

    Article Title: Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

    doi: 10.1085/jgp.200910355

    Figure Lengend Snippet: cAMP does not affect granule mobility. Before each experiment, cells were preincubated with 1 μM ionomycin for at least 5 min in Ca 2+ -free solution (0 Ca 2+ ), and all test solutions contained 1 μM ionomycin in a Ca 2+ -free solution. Average speed of granule movement during treatment with (A) 2 mM Ca 2+ (‘2 Ca 2+ ’, n = 2, n = 19), (B) 1 μM FSK ( n = 3, n = 30), and (C) 2 mM Ca 2+ with FSK ( n = 3, n = 25), where N and n indicate the number of cells and granules for each experiment. External Ca 2+ concentration was exchanged from 0 to 2 mM Ca 2+ to increase [Ca 2+ ] i . Red dotted lines indicate average granule speed in control conditions before external Ca 2+ or FSK treatments. Average MSD of the same granules is plotted on the right side of each figure. The same color coding for the treatments is used.

    Article Snippet: UTP, ionomycin, H-89, and Rp-8-Br-cAMPS were obtained from Calbiochem.

    Techniques: Concentration Assay

    Nuclear ErbB2 increases protein synthesis and cell size. A, left, Total protein contents from equal cell numbers of MCF-7 and HER18 cells were determined by the Bradford method. Right, 35 S-methionine labeling of protein synthesis in equal cell numbers of MCF-7 and HER18 cells were measured by scintillation counting. B, Knockdown of ErbB2 using siRNA results in decreased protein biosynthesis. Cells transfected with ErbB2 or non-specific (NS) siRNAs were monitored for total protein concentration (left, top) and 35 S-methionine incorporation (right, top) as analyzed in A. ErbB2 protein knockdown was confirmed by western blotting (bottom). C and D, Total protein level (C) and cell size (D) are increased in wild-type (WT) ErbB-2-expressing cells but not in cells expressing ErbB-2Δ NLS mutant. MCF-7 and MDA-MB-231 cells expressing WT ErbB-2, ErbB-2Δ NLS mutant or vector control were assayed for total protein synthesis (C, top) and ErbB2 protein expression (C, bottom) as in B. Cells were also analyzed by flow cytometry to detect DNA content and cell size of G1-, S- and G2/M- phase cells using the parameter mean forward scatter height (FSC-H), which is a measure of relative cell size (D). P value was analyzed by Student’s t test.

    Journal: Cancer research

    Article Title: Nuclear ErbB-2 Enhances Translation and Cell Growth by activating transcription of rRNA genes

    doi: 10.1158/0008-5472.CAN-10-3504

    Figure Lengend Snippet: Nuclear ErbB2 increases protein synthesis and cell size. A, left, Total protein contents from equal cell numbers of MCF-7 and HER18 cells were determined by the Bradford method. Right, 35 S-methionine labeling of protein synthesis in equal cell numbers of MCF-7 and HER18 cells were measured by scintillation counting. B, Knockdown of ErbB2 using siRNA results in decreased protein biosynthesis. Cells transfected with ErbB2 or non-specific (NS) siRNAs were monitored for total protein concentration (left, top) and 35 S-methionine incorporation (right, top) as analyzed in A. ErbB2 protein knockdown was confirmed by western blotting (bottom). C and D, Total protein level (C) and cell size (D) are increased in wild-type (WT) ErbB-2-expressing cells but not in cells expressing ErbB-2Δ NLS mutant. MCF-7 and MDA-MB-231 cells expressing WT ErbB-2, ErbB-2Δ NLS mutant or vector control were assayed for total protein synthesis (C, top) and ErbB2 protein expression (C, bottom) as in B. Cells were also analyzed by flow cytometry to detect DNA content and cell size of G1-, S- and G2/M- phase cells using the parameter mean forward scatter height (FSC-H), which is a measure of relative cell size (D). P value was analyzed by Student’s t test.

    Article Snippet: The antibodies and chemicals used were: anti-ErbB-2 (Calbiochem, Thermo Scientific); anti-β-actin, anti-α-tubulin and mouse IgG (Sigma); anti-RPA194 (Santa Cruz); anti-Lamin B (Calbiochem); anti-BrdU (Molecular Probes, Abcam); anti-Akt, anti-phospho-Akt, anti-ERK, anti-phospho-ERK and U0126 (Cell Signaling); Br-UTP, α-amanitin and actinomycin D (Sigma); (Promega) and Heregulin (Thermo Scientific).

    Techniques: Labeling, Transfection, Protein Concentration, Western Blot, Expressing, Mutagenesis, Multiple Displacement Amplification, Plasmid Preparation, Flow Cytometry, Cytometry

    Model depicting the novel function of nuclear ErbB-2 in regulation of RNA Pol I-mediated rRNA synthesis.

    Journal: Cancer research

    Article Title: Nuclear ErbB-2 Enhances Translation and Cell Growth by activating transcription of rRNA genes

    doi: 10.1158/0008-5472.CAN-10-3504

    Figure Lengend Snippet: Model depicting the novel function of nuclear ErbB-2 in regulation of RNA Pol I-mediated rRNA synthesis.

    Article Snippet: The antibodies and chemicals used were: anti-ErbB-2 (Calbiochem, Thermo Scientific); anti-β-actin, anti-α-tubulin and mouse IgG (Sigma); anti-RPA194 (Santa Cruz); anti-Lamin B (Calbiochem); anti-BrdU (Molecular Probes, Abcam); anti-Akt, anti-phospho-Akt, anti-ERK, anti-phospho-ERK and U0126 (Cell Signaling); Br-UTP, α-amanitin and actinomycin D (Sigma); (Promega) and Heregulin (Thermo Scientific).

    Techniques:

    A, ErbB2 associates with RNA Pol I. Total lysate (left) and cytoplasmic and nuclear lysates (right) were IP and analyzed by western blotting with indicated antibodies. B, left, Confocal microscopy of ErbB2 (red) and RNA Pol I (green) colocalization in the nucleus (blue). Inset is enlarged. Scale bar, 5 µm. Right, Electron microscopy showing colocalization of ErbB-2 (solid arrows, 5 nm gold-labeled) and RNA Pol I (arrowheads, 1 nm gold-labeled) in the nucleus. Insets are enlarged. Cy, cytoplasm; Nu, nucleus. Negative control without primary antibody staining; gold particles were not detected even with gold particle-labeled secondary antibodies (lower panels). C, top, ErbB2 forms complexes with RNA Pol I and β-actin. Cell lysates were primarily IP (1st IP) with anti-ErbB2 or rabbit IgG (rIgG) antibodies, secondarily IP (2nd IP) with antibodies against RNA Pol I or mIgG and analyzed by Western blotting. Bottom, Electron microscopy shows colocalization of ErbB2 (thin arrows, 25 nm gold-labeled), β-actin (arrowheads, 10 nm gold-labeled) and RNA Pol I (thick arrows, 6 nm gold-labeled) in the nucleus. Insets are enlarged. Negative control as in B (right). D, Total cell lysates from cells transfected with β-actin siRNA or non-specific siRNA control (NS) were IP with RNA Pol I and subjected to western blotting as indicated. E, Protein extracts from ErbB-2 siRNA- or NS siRNA-transfected cells were IP with β-actin and analyzed as in (D).

    Journal: Cancer research

    Article Title: Nuclear ErbB-2 Enhances Translation and Cell Growth by activating transcription of rRNA genes

    doi: 10.1158/0008-5472.CAN-10-3504

    Figure Lengend Snippet: A, ErbB2 associates with RNA Pol I. Total lysate (left) and cytoplasmic and nuclear lysates (right) were IP and analyzed by western blotting with indicated antibodies. B, left, Confocal microscopy of ErbB2 (red) and RNA Pol I (green) colocalization in the nucleus (blue). Inset is enlarged. Scale bar, 5 µm. Right, Electron microscopy showing colocalization of ErbB-2 (solid arrows, 5 nm gold-labeled) and RNA Pol I (arrowheads, 1 nm gold-labeled) in the nucleus. Insets are enlarged. Cy, cytoplasm; Nu, nucleus. Negative control without primary antibody staining; gold particles were not detected even with gold particle-labeled secondary antibodies (lower panels). C, top, ErbB2 forms complexes with RNA Pol I and β-actin. Cell lysates were primarily IP (1st IP) with anti-ErbB2 or rabbit IgG (rIgG) antibodies, secondarily IP (2nd IP) with antibodies against RNA Pol I or mIgG and analyzed by Western blotting. Bottom, Electron microscopy shows colocalization of ErbB2 (thin arrows, 25 nm gold-labeled), β-actin (arrowheads, 10 nm gold-labeled) and RNA Pol I (thick arrows, 6 nm gold-labeled) in the nucleus. Insets are enlarged. Negative control as in B (right). D, Total cell lysates from cells transfected with β-actin siRNA or non-specific siRNA control (NS) were IP with RNA Pol I and subjected to western blotting as indicated. E, Protein extracts from ErbB-2 siRNA- or NS siRNA-transfected cells were IP with β-actin and analyzed as in (D).

    Article Snippet: The antibodies and chemicals used were: anti-ErbB-2 (Calbiochem, Thermo Scientific); anti-β-actin, anti-α-tubulin and mouse IgG (Sigma); anti-RPA194 (Santa Cruz); anti-Lamin B (Calbiochem); anti-BrdU (Molecular Probes, Abcam); anti-Akt, anti-phospho-Akt, anti-ERK, anti-phospho-ERK and U0126 (Cell Signaling); Br-UTP, α-amanitin and actinomycin D (Sigma); (Promega) and Heregulin (Thermo Scientific).

    Techniques: Western Blot, Confocal Microscopy, Electron Microscopy, Labeling, Negative Control, Staining, Transfection

    Nuclear ErbB2 increases RNA Pol I transcription in vivo. A, Cells transfected with ErbB2 or non-specific (NS) siRNAs were assessed for 45S pre-rRNA synthesis by RT-qPCR (top) and ErbB2 protein expression (bottom). error bar, SD; B, SKBR3 and HER18 cells transfected with ErbB2 siRNA or NS siRNA were subjected to Br-UTP incorporation assays of nascent nucleolar RNA and confocal microscopy for ErbB-2 (green), Br-UTP (red) and nuclei (DAPI, blue). Representative images are from SKBR3 cells. Percent of Br-UTP positive cells shown as means with SD. C, Permeabilized SKBR3 cells were incubated with Br-UTP to label active transcription sites with or without α-amanitin. Confocal microscopy was as in B. D, left, Cells were examined for 45S pre-rRNA synthesis (top) and ErbB2 protein (bottom) as in A. Relative amounts of 45S pre-rRNA shown as means with SD. Middle, Br-UTP incorporation assays of nascent nucleolar RNA in MCF-7 and the MCF-7 stable cell line expressing wild-type ErbB2 (HER18). Percent of Br-UTP positive cells shown as means with SD. Right, Cells transfected with increasing amounts of ErbB2 were measured for 45S pre-rRNA synthesis (top) and ErbB2 protein (middle) as in A, and RT-PCR of pre-rRNA and internal control GAPDH (bottom). E, Transient (293) or stable (MCF-7 and MDA-MB 231) transfectants of wild-type ErbB-2 (WT), ErbB-2Δ NLS mutant or vector (Vec) were assayed for co-IP of ErbB-2 with RNA Pol I (left), 45S pre-rRNA synthesis and ErbB2 protein (right) as in D (right).

    Journal: Cancer research

    Article Title: Nuclear ErbB-2 Enhances Translation and Cell Growth by activating transcription of rRNA genes

    doi: 10.1158/0008-5472.CAN-10-3504

    Figure Lengend Snippet: Nuclear ErbB2 increases RNA Pol I transcription in vivo. A, Cells transfected with ErbB2 or non-specific (NS) siRNAs were assessed for 45S pre-rRNA synthesis by RT-qPCR (top) and ErbB2 protein expression (bottom). error bar, SD; B, SKBR3 and HER18 cells transfected with ErbB2 siRNA or NS siRNA were subjected to Br-UTP incorporation assays of nascent nucleolar RNA and confocal microscopy for ErbB-2 (green), Br-UTP (red) and nuclei (DAPI, blue). Representative images are from SKBR3 cells. Percent of Br-UTP positive cells shown as means with SD. C, Permeabilized SKBR3 cells were incubated with Br-UTP to label active transcription sites with or without α-amanitin. Confocal microscopy was as in B. D, left, Cells were examined for 45S pre-rRNA synthesis (top) and ErbB2 protein (bottom) as in A. Relative amounts of 45S pre-rRNA shown as means with SD. Middle, Br-UTP incorporation assays of nascent nucleolar RNA in MCF-7 and the MCF-7 stable cell line expressing wild-type ErbB2 (HER18). Percent of Br-UTP positive cells shown as means with SD. Right, Cells transfected with increasing amounts of ErbB2 were measured for 45S pre-rRNA synthesis (top) and ErbB2 protein (middle) as in A, and RT-PCR of pre-rRNA and internal control GAPDH (bottom). E, Transient (293) or stable (MCF-7 and MDA-MB 231) transfectants of wild-type ErbB-2 (WT), ErbB-2Δ NLS mutant or vector (Vec) were assayed for co-IP of ErbB-2 with RNA Pol I (left), 45S pre-rRNA synthesis and ErbB2 protein (right) as in D (right).

    Article Snippet: The antibodies and chemicals used were: anti-ErbB-2 (Calbiochem, Thermo Scientific); anti-β-actin, anti-α-tubulin and mouse IgG (Sigma); anti-RPA194 (Santa Cruz); anti-Lamin B (Calbiochem); anti-BrdU (Molecular Probes, Abcam); anti-Akt, anti-phospho-Akt, anti-ERK, anti-phospho-ERK and U0126 (Cell Signaling); Br-UTP, α-amanitin and actinomycin D (Sigma); (Promega) and Heregulin (Thermo Scientific).

    Techniques: In Vivo, Transfection, Quantitative RT-PCR, Expressing, Confocal Microscopy, Incubation, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Multiple Displacement Amplification, Mutagenesis, Plasmid Preparation, Co-Immunoprecipitation Assay

    ErbB-2 enhances binding of RNA Pol I to rDNA and associates with rDNA during RNA Pol I transcription in vivo. A, left, ChIP with RNA Pol I antibody or mIgG of crosslinked chromatin from MCF-7 and HER18 cells. The DNA was amplified by PCR (top) or quantitative PCR (bottom) using primers specific to the 5’-terminal region of human rDNA or the 28S rRNA coding sequence. The relative rDNA occupancy of RNA Pol I was determined by normalizing rDNA in the Pol I ChIP with rDNA in the mIgG ChIP, then comparing with that in the input chromatin and shown as mean with SD. p value was analyzed by Student’s t test. Western blot shows protein expression. Right, ChIPs from cells transfected with ErbB-2 (+) or non-specific (−) siRNAs using RNA Pol I or mIgG antibodies were analyzed as in left. B, Crosslinked chromatin from ErbB-2-expressing cell lines was immunoprecipitated with antibodies against ErbB-2 or mIgG and analyzed as in A. C, Sequential ChIP assays were performed with first immunoprecipitation (1st ChIP) using anti-ErbB2 or mIgG and second immunoprecipitation (2nd ChIP) using antibodies against β-actin, RNA Pol I or mIgG. Chromatin samples were amplified as in B. D, left, ChIPs from cells transfected with β-actin or non-specific (NS) siRNAs using ErbB-2 antibody were analyzed as in B. Right, Knockdown of β-actin. E, ErbB-2-expressing cells were treated with (+) or without (−) RNA Pol I inhibitor, actinomycin D (Act. D). pre-rRNA synthesis (left) and rDNA occupancy of ErbB-2, RNA Pol I and β-actin (right) were analyzed using RT-qPCR and ChIP assays, respectively. Bar diagram shows relative rDNA binding levels of ErbB-2, RNA Pol I and βactin in Act. D-treated cells compared with that in cells without treatment.

    Journal: Cancer research

    Article Title: Nuclear ErbB-2 Enhances Translation and Cell Growth by activating transcription of rRNA genes

    doi: 10.1158/0008-5472.CAN-10-3504

    Figure Lengend Snippet: ErbB-2 enhances binding of RNA Pol I to rDNA and associates with rDNA during RNA Pol I transcription in vivo. A, left, ChIP with RNA Pol I antibody or mIgG of crosslinked chromatin from MCF-7 and HER18 cells. The DNA was amplified by PCR (top) or quantitative PCR (bottom) using primers specific to the 5’-terminal region of human rDNA or the 28S rRNA coding sequence. The relative rDNA occupancy of RNA Pol I was determined by normalizing rDNA in the Pol I ChIP with rDNA in the mIgG ChIP, then comparing with that in the input chromatin and shown as mean with SD. p value was analyzed by Student’s t test. Western blot shows protein expression. Right, ChIPs from cells transfected with ErbB-2 (+) or non-specific (−) siRNAs using RNA Pol I or mIgG antibodies were analyzed as in left. B, Crosslinked chromatin from ErbB-2-expressing cell lines was immunoprecipitated with antibodies against ErbB-2 or mIgG and analyzed as in A. C, Sequential ChIP assays were performed with first immunoprecipitation (1st ChIP) using anti-ErbB2 or mIgG and second immunoprecipitation (2nd ChIP) using antibodies against β-actin, RNA Pol I or mIgG. Chromatin samples were amplified as in B. D, left, ChIPs from cells transfected with β-actin or non-specific (NS) siRNAs using ErbB-2 antibody were analyzed as in B. Right, Knockdown of β-actin. E, ErbB-2-expressing cells were treated with (+) or without (−) RNA Pol I inhibitor, actinomycin D (Act. D). pre-rRNA synthesis (left) and rDNA occupancy of ErbB-2, RNA Pol I and β-actin (right) were analyzed using RT-qPCR and ChIP assays, respectively. Bar diagram shows relative rDNA binding levels of ErbB-2, RNA Pol I and βactin in Act. D-treated cells compared with that in cells without treatment.

    Article Snippet: The antibodies and chemicals used were: anti-ErbB-2 (Calbiochem, Thermo Scientific); anti-β-actin, anti-α-tubulin and mouse IgG (Sigma); anti-RPA194 (Santa Cruz); anti-Lamin B (Calbiochem); anti-BrdU (Molecular Probes, Abcam); anti-Akt, anti-phospho-Akt, anti-ERK, anti-phospho-ERK and U0126 (Cell Signaling); Br-UTP, α-amanitin and actinomycin D (Sigma); (Promega) and Heregulin (Thermo Scientific).

    Techniques: Binding Assay, In Vivo, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Sequencing, Western Blot, Expressing, Transfection, Immunoprecipitation, Activated Clotting Time Assay, Quantitative RT-PCR

    for details). The middle plan 18 within the nucleus is presented of a particular group of ErbB-2 (green)-βactin (red) complexes whose colocalization is indicated by white arrows in the merged images. Scale bar, 5 µm.

    Journal: Cancer research

    Article Title: Nuclear ErbB-2 Enhances Translation and Cell Growth by activating transcription of rRNA genes

    doi: 10.1158/0008-5472.CAN-10-3504

    Figure Lengend Snippet: for details). The middle plan 18 within the nucleus is presented of a particular group of ErbB-2 (green)-βactin (red) complexes whose colocalization is indicated by white arrows in the merged images. Scale bar, 5 µm.

    Article Snippet: The antibodies and chemicals used were: anti-ErbB-2 (Calbiochem, Thermo Scientific); anti-β-actin, anti-α-tubulin and mouse IgG (Sigma); anti-RPA194 (Santa Cruz); anti-Lamin B (Calbiochem); anti-BrdU (Molecular Probes, Abcam); anti-Akt, anti-phospho-Akt, anti-ERK, anti-phospho-ERK and U0126 (Cell Signaling); Br-UTP, α-amanitin and actinomycin D (Sigma); (Promega) and Heregulin (Thermo Scientific).

    Techniques: