oligo dt primers  (Thermo Fisher)


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    Name:
    Oligo dT Primer 50 µM
    Description:
    These are the same Ambion primers that are currently included in the RETROscript Kit SKU AM1710 They are provided at a stock concentration of 50 µM and are functionally tested using the RETROscript Kit
    Catalog Number:
    am5730g
    Price:
    None
    Applications:
    PCR & Real-Time PCR|RT-PCR|Reverse Transcription|Two-Step RT-PCR
    Category:
    Oligos Primers Probes Nucleotides
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    Structured Review

    Thermo Fisher oligo dt primers
    Ferroportin is expressed in hPASMCs and regulated by hepcidin. Confluent hPASMCs were ( A ) either mock-treated or treated with 1 µg/mL hepcidin for 2.5 h and total <t>RNA</t> extracted using RNeasy kit, cDNA synthesised using <t>oligo-dT</t> primers and RT-PCR performed using SYBR green with human Fpn (ferroportin) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) mock-treated for 24 h, cells lysed and total protein extracted, quantified by Bradford assay and 40 µg of protein separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blotting was performed using Rabbit anti-Fpn as primary and Goat anti-rabbit IgG conjugated with horse-radish peroxidase as secondary antibodies. Human intestinal lysates (Abcam) were used as positive control. N = 3 ( C ) either mock-treated or treated with 1 µg/mL hepcidin for 24 h, cells lysed and total protein extracted. Fpn expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 1 µg/mL hepcidin for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm; N = 5. Student’s t test was performed; **p
    These are the same Ambion primers that are currently included in the RETROscript Kit SKU AM1710 They are provided at a stock concentration of 50 µM and are functionally tested using the RETROscript Kit
    https://www.bioz.com/result/oligo dt primers/product/Thermo Fisher
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    Images

    1) Product Images from "The Hepcidin/Ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells"

    Article Title: The Hepcidin/Ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31095-0

    Ferroportin is expressed in hPASMCs and regulated by hepcidin. Confluent hPASMCs were ( A ) either mock-treated or treated with 1 µg/mL hepcidin for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human Fpn (ferroportin) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) mock-treated for 24 h, cells lysed and total protein extracted, quantified by Bradford assay and 40 µg of protein separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blotting was performed using Rabbit anti-Fpn as primary and Goat anti-rabbit IgG conjugated with horse-radish peroxidase as secondary antibodies. Human intestinal lysates (Abcam) were used as positive control. N = 3 ( C ) either mock-treated or treated with 1 µg/mL hepcidin for 24 h, cells lysed and total protein extracted. Fpn expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 1 µg/mL hepcidin for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm; N = 5. Student’s t test was performed; **p
    Figure Legend Snippet: Ferroportin is expressed in hPASMCs and regulated by hepcidin. Confluent hPASMCs were ( A ) either mock-treated or treated with 1 µg/mL hepcidin for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human Fpn (ferroportin) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) mock-treated for 24 h, cells lysed and total protein extracted, quantified by Bradford assay and 40 µg of protein separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blotting was performed using Rabbit anti-Fpn as primary and Goat anti-rabbit IgG conjugated with horse-radish peroxidase as secondary antibodies. Human intestinal lysates (Abcam) were used as positive control. N = 3 ( C ) either mock-treated or treated with 1 µg/mL hepcidin for 24 h, cells lysed and total protein extracted. Fpn expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 1 µg/mL hepcidin for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm; N = 5. Student’s t test was performed; **p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Bradford Assay, SDS Page, Western Blot, Positive Control, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Microscopy

    IL-6 increased hepcidin expression and down-regulated ferroportin in hPASMCs. Confluent hPASMCs were either mock-treated or treated with 10 ng/mL IL-6 ( A ) for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human hepcidin (Hamp-1) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) for 24 h, media supernatant collected and hepcidin secretion was quantitated using an ELISA kit (R D Systems). N = 4 ( C ) for 24 h cells lysed and total protein extracted. Ferroportin expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 10 ng/mL IL-6 for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm, N = 5. Student’s t test was performed; **p
    Figure Legend Snippet: IL-6 increased hepcidin expression and down-regulated ferroportin in hPASMCs. Confluent hPASMCs were either mock-treated or treated with 10 ng/mL IL-6 ( A ) for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human hepcidin (Hamp-1) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) for 24 h, media supernatant collected and hepcidin secretion was quantitated using an ELISA kit (R D Systems). N = 4 ( C ) for 24 h cells lysed and total protein extracted. Ferroportin expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 10 ng/mL IL-6 for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm, N = 5. Student’s t test was performed; **p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy

    2) Product Images from "Ibudilast, a Pharmacologic Phosphodiesterase Inhibitor, Prevents Human Immunodeficiency Virus-1 Tat-Mediated Activation of Microglial Cells"

    Article Title: Ibudilast, a Pharmacologic Phosphodiesterase Inhibitor, Prevents Human Immunodeficiency Virus-1 Tat-Mediated Activation of Microglial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018633

    Ibudilast inhibits Tat-induced TNFα transcript levels. BV-2 cells (2.5×10 5 ) were left untreated (NT) or were treated with Tat (100 nM) for 4 h with or without pre-treatment for 30 min. with increasing concentrations of ibudilast (Ib) or vehicle (Veh). Total RNA was collected, reverse transcribed using oligo-dT primers, and subjected to Real-Time SYBR Green RT-PCR amplification. Fold induction of TNFα mRNA species was normalized to those of GAPDH and presented as a function of the expression level in NT samples. Data represent mean ± SEM of values derived from three replicates from a single representative experiment. Statistical significance (***, p
    Figure Legend Snippet: Ibudilast inhibits Tat-induced TNFα transcript levels. BV-2 cells (2.5×10 5 ) were left untreated (NT) or were treated with Tat (100 nM) for 4 h with or without pre-treatment for 30 min. with increasing concentrations of ibudilast (Ib) or vehicle (Veh). Total RNA was collected, reverse transcribed using oligo-dT primers, and subjected to Real-Time SYBR Green RT-PCR amplification. Fold induction of TNFα mRNA species was normalized to those of GAPDH and presented as a function of the expression level in NT samples. Data represent mean ± SEM of values derived from three replicates from a single representative experiment. Statistical significance (***, p

    Techniques Used: SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, Derivative Assay

    3) Product Images from "The Hepcidin/Ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells"

    Article Title: The Hepcidin/Ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31095-0

    Ferroportin is expressed in hPASMCs and regulated by hepcidin. Confluent hPASMCs were ( A ) either mock-treated or treated with 1 µg/mL hepcidin for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human Fpn (ferroportin) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) mock-treated for 24 h, cells lysed and total protein extracted, quantified by Bradford assay and 40 µg of protein separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blotting was performed using Rabbit anti-Fpn as primary and Goat anti-rabbit IgG conjugated with horse-radish peroxidase as secondary antibodies. Human intestinal lysates (Abcam) were used as positive control. N = 3 ( C ) either mock-treated or treated with 1 µg/mL hepcidin for 24 h, cells lysed and total protein extracted. Fpn expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 1 µg/mL hepcidin for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm; N = 5. Student’s t test was performed; **p
    Figure Legend Snippet: Ferroportin is expressed in hPASMCs and regulated by hepcidin. Confluent hPASMCs were ( A ) either mock-treated or treated with 1 µg/mL hepcidin for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human Fpn (ferroportin) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) mock-treated for 24 h, cells lysed and total protein extracted, quantified by Bradford assay and 40 µg of protein separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blotting was performed using Rabbit anti-Fpn as primary and Goat anti-rabbit IgG conjugated with horse-radish peroxidase as secondary antibodies. Human intestinal lysates (Abcam) were used as positive control. N = 3 ( C ) either mock-treated or treated with 1 µg/mL hepcidin for 24 h, cells lysed and total protein extracted. Fpn expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 1 µg/mL hepcidin for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm; N = 5. Student’s t test was performed; **p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Bradford Assay, SDS Page, Western Blot, Positive Control, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Microscopy

    IL-6 increased hepcidin expression and down-regulated ferroportin in hPASMCs. Confluent hPASMCs were either mock-treated or treated with 10 ng/mL IL-6 ( A ) for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human hepcidin (Hamp-1) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) for 24 h, media supernatant collected and hepcidin secretion was quantitated using an ELISA kit (R D Systems). N = 4 ( C ) for 24 h cells lysed and total protein extracted. Ferroportin expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 10 ng/mL IL-6 for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm, N = 5. Student’s t test was performed; **p
    Figure Legend Snippet: IL-6 increased hepcidin expression and down-regulated ferroportin in hPASMCs. Confluent hPASMCs were either mock-treated or treated with 10 ng/mL IL-6 ( A ) for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human hepcidin (Hamp-1) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) for 24 h, media supernatant collected and hepcidin secretion was quantitated using an ELISA kit (R D Systems). N = 4 ( C ) for 24 h cells lysed and total protein extracted. Ferroportin expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 10 ng/mL IL-6 for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm, N = 5. Student’s t test was performed; **p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy

    4) Product Images from "RRP7A links primary microcephaly to radial glial cells and dysfunction of ribosomal biogenesis, neurogenesis and ciliary resorption"

    Article Title: RRP7A links primary microcephaly to radial glial cells and dysfunction of ribosomal biogenesis, neurogenesis and ciliary resorption

    Journal: bioRxiv

    doi: 10.1101/793877

    RRP7A localizes to nucleoli, centrosomes and primary cilia, and nucleolar localization and rRNA processing are disrupted in patient HDFs. a , IFM analysis on the localization of RRP7A ( green ) to nucleoli (open arrows, light microscopy (LM)), the primary cilium (closed arrows, ARL13B, red ) in serum-depleted cultures of control human dermal fibroblasts (HDF). The nucleus (nu) is stained with DAPI ( blue ). Scale bar, 20 µm. Insert scale bar, Scale bar, 5 µm. b , Zoomed image of the cilium-centrosome axis depicted in (a) with the ciliary base/centrosome marked with anti-Pericentrin-2 (asterisk, PCTN-2, magenta ). Scale bar, 5 µm. c , IFM analysis of localization of RRP7A ( red ) to nucleoli (open arrows) in serum supplemented cultures. Scale bar, 20 µm. Scale bar in zoomed image, 5 µm. d , IFM analysis on the localization of RRP7A ( green ) to nucleoli (stippled-lined areas) and the primary cilium (ARL13B, red ) in serum-depleted cultures of control HDFs (left panel) and patient HDFs (right panel). Scale bars, 10 µm. The insert shows magnification of boxed areas with shifted overlays between RRP7A ( green closed arrow) and ARL13B ( red closed arrow). The nucleus (nu) is stained with DAPI. Scale bars, 2 µm. e , Quantification of the relative levels of RRP7A in nucleoli in control and patient HDFs shown in (d) (n=5). f , Quantification of the relative levels of RRP7A in nucleoli at the cilium-centrosome axis in control and patient HDFs shown in (d) (n=4). g , Map of the human pre-rRNA transcript with annotated processing sites and a simplified outline of the two main processing pathways with short-lived precursors in grey. In pathway 1, cleavage of 45S occurs at sites A0 and 1 to produce 41S that is subsequently split into two parts by cleavage at site 2 to yield the direct precursors for the small and large subunit rRNAs, respectively. In pathway 2, this order is reversed, such that 45S is first cleaved at site 2 yielding precursors for the small and large subunit rRNAs. The target sites of oligo nucleotide probes a and b are indicated on the map. h , Northern blots of parallel gel runs of total RNA samples from control and patient HDFs with mutations in RRP7A (patient) and WDR62 (MCPH2 pt.), respectively. Black arrows indicate processing intermediates inferred from the analyses and grey arrows mark the migration of mature rRNA species as inferred from re-probing of the filters with probes targeting these RNA species.
    Figure Legend Snippet: RRP7A localizes to nucleoli, centrosomes and primary cilia, and nucleolar localization and rRNA processing are disrupted in patient HDFs. a , IFM analysis on the localization of RRP7A ( green ) to nucleoli (open arrows, light microscopy (LM)), the primary cilium (closed arrows, ARL13B, red ) in serum-depleted cultures of control human dermal fibroblasts (HDF). The nucleus (nu) is stained with DAPI ( blue ). Scale bar, 20 µm. Insert scale bar, Scale bar, 5 µm. b , Zoomed image of the cilium-centrosome axis depicted in (a) with the ciliary base/centrosome marked with anti-Pericentrin-2 (asterisk, PCTN-2, magenta ). Scale bar, 5 µm. c , IFM analysis of localization of RRP7A ( red ) to nucleoli (open arrows) in serum supplemented cultures. Scale bar, 20 µm. Scale bar in zoomed image, 5 µm. d , IFM analysis on the localization of RRP7A ( green ) to nucleoli (stippled-lined areas) and the primary cilium (ARL13B, red ) in serum-depleted cultures of control HDFs (left panel) and patient HDFs (right panel). Scale bars, 10 µm. The insert shows magnification of boxed areas with shifted overlays between RRP7A ( green closed arrow) and ARL13B ( red closed arrow). The nucleus (nu) is stained with DAPI. Scale bars, 2 µm. e , Quantification of the relative levels of RRP7A in nucleoli in control and patient HDFs shown in (d) (n=5). f , Quantification of the relative levels of RRP7A in nucleoli at the cilium-centrosome axis in control and patient HDFs shown in (d) (n=4). g , Map of the human pre-rRNA transcript with annotated processing sites and a simplified outline of the two main processing pathways with short-lived precursors in grey. In pathway 1, cleavage of 45S occurs at sites A0 and 1 to produce 41S that is subsequently split into two parts by cleavage at site 2 to yield the direct precursors for the small and large subunit rRNAs, respectively. In pathway 2, this order is reversed, such that 45S is first cleaved at site 2 yielding precursors for the small and large subunit rRNAs. The target sites of oligo nucleotide probes a and b are indicated on the map. h , Northern blots of parallel gel runs of total RNA samples from control and patient HDFs with mutations in RRP7A (patient) and WDR62 (MCPH2 pt.), respectively. Black arrows indicate processing intermediates inferred from the analyses and grey arrows mark the migration of mature rRNA species as inferred from re-probing of the filters with probes targeting these RNA species.

    Techniques Used: Light Microscopy, Staining, Northern Blot, Migration

    5) Product Images from "Nuclear Factor-Kappa B Family Member RelB Inhibits Human Immunodeficiency Virus-1 Tat-Induced Tumor Necrosis Factor-Alpha Production"

    Article Title: Nuclear Factor-Kappa B Family Member RelB Inhibits Human Immunodeficiency Virus-1 Tat-Induced Tumor Necrosis Factor-Alpha Production

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011875

    Transgenic expression of Tat induces RelB synthesis in mouse brain. 6-week old Tat-transgenic mice were induced with doxycycline for the indicated periods of time. Total RNA was extracted from brain tissue, reverse transcribed using oligo-dT primers, and subjected to Real-Time SYBR Green RT-PCR amplification. Fold induction of Tat, RelB and RelA mRNA species was normalized to GAPDH and presented as a function of the expression level in D0 samples. Data represent mean ± SEM of four replicates for D0 and D14 samples, and five replicates for D3 samples. Statistical significance (***, p
    Figure Legend Snippet: Transgenic expression of Tat induces RelB synthesis in mouse brain. 6-week old Tat-transgenic mice were induced with doxycycline for the indicated periods of time. Total RNA was extracted from brain tissue, reverse transcribed using oligo-dT primers, and subjected to Real-Time SYBR Green RT-PCR amplification. Fold induction of Tat, RelB and RelA mRNA species was normalized to GAPDH and presented as a function of the expression level in D0 samples. Data represent mean ± SEM of four replicates for D0 and D14 samples, and five replicates for D3 samples. Statistical significance (***, p

    Techniques Used: Transgenic Assay, Expressing, Mouse Assay, SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction, Amplification

    6) Product Images from "Midkine is a NF-?B-inducible gene that supports prostate cancer cell survival"

    Article Title: Midkine is a NF-?B-inducible gene that supports prostate cancer cell survival

    Journal: BMC Medical Genomics

    doi: 10.1186/1755-8794-1-6

    Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P
    Figure Legend Snippet: Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P

    Techniques Used: Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

    7) Product Images from "Gamma secretase dependent release of the CD44 cytoplasmic tail upregulates IFI16 in cd44-/- tumor cells, MEFs and macrophages"

    Article Title: Gamma secretase dependent release of the CD44 cytoplasmic tail upregulates IFI16 in cd44-/- tumor cells, MEFs and macrophages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207358

    Impaired induction of IFI16 expression in cd44 -/- bone marrow-derived macrophages. Bone marrow-derived macrophages (BMDMs) from wild type and cd44 -/- mice were differentiated as described in Materials and Methods. Subsequently, the macrophages were serum-starved in macrophage medium w/o heat-inactivated FBS for 24 h and stimulated with 10 ng/ml IFN-γ for 6 h (A and B) or transfected with the VACV70mer oligo (C and D). RNA was determined as in Fig 4 . Experiments were done in triplicates. For statistics see Materials and methods .
    Figure Legend Snippet: Impaired induction of IFI16 expression in cd44 -/- bone marrow-derived macrophages. Bone marrow-derived macrophages (BMDMs) from wild type and cd44 -/- mice were differentiated as described in Materials and Methods. Subsequently, the macrophages were serum-starved in macrophage medium w/o heat-inactivated FBS for 24 h and stimulated with 10 ng/ml IFN-γ for 6 h (A and B) or transfected with the VACV70mer oligo (C and D). RNA was determined as in Fig 4 . Experiments were done in triplicates. For statistics see Materials and methods .

    Techniques Used: Expressing, Derivative Assay, Mouse Assay, Transfection

    Rescue of viral DNA-induced IFI16 and IFN-β expression by CD44-ICD in cd44 -/- MEFs. MEFs from CD44 complete knockout mice were lentivirally transduced with either CD44 full length or CD44-ICD as described in Materials and Methods. Two days after transduction cd44+/+ and cd44-/- MEFs were transfected with the VACV70mer oligo for 6 h as described in Materials and Methods. RNA was isolated and transcribed into cDNA. cDNA was analysed by RT-qPCR using primers for murine IFI16 (primers 11 and 12, Table 1 ) and IFN-β (primers 13 and 14, Table 1 ) and actin for normalization (primers 7 and 8, Table 1 ). Experiments were done in triplicates. For statistics see Materials and methods .
    Figure Legend Snippet: Rescue of viral DNA-induced IFI16 and IFN-β expression by CD44-ICD in cd44 -/- MEFs. MEFs from CD44 complete knockout mice were lentivirally transduced with either CD44 full length or CD44-ICD as described in Materials and Methods. Two days after transduction cd44+/+ and cd44-/- MEFs were transfected with the VACV70mer oligo for 6 h as described in Materials and Methods. RNA was isolated and transcribed into cDNA. cDNA was analysed by RT-qPCR using primers for murine IFI16 (primers 11 and 12, Table 1 ) and IFN-β (primers 13 and 14, Table 1 ) and actin for normalization (primers 7 and 8, Table 1 ). Experiments were done in triplicates. For statistics see Materials and methods .

    Techniques Used: Expressing, Knock-Out, Mouse Assay, Transduction, Transfection, Isolation, Quantitative RT-PCR

    8) Product Images from "Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication"

    Article Title: Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-183

    FHV infection and replicon expression upregulate partially overlapping sets of Drosophila genes . (A) Venn diagram indicating the number of upregulated genes unique to FHV-infected cells (white circle), unique to FHV replicon-expressing cells (grey circle), or upregulated with both (black convergence). Total numbers of genes are given within the indicated regions, and complete lists and descriptions of genes are provided in Table 1 and as Additional Files 1 , 2 and 3 . Specific upregulated genes involved in lipid metabolism, as identified by GO terms, are shown by either their gene symbols or CG designations. (B) Semi-quantitative RT-PCR validation of Cct1 and Cct2 mRNA upregulation in Drosophila S2 cells infected with FHV. Decreasing amounts of cDNA generated by RT with oligo-dT primers and equivalent amounts of total RNA from mock (upper gel) or FHV-infected S2 cells (lower gel) were amplified with gene-specific primers for Drosophila actin ( Act5C) , Cct1 , or Cct2 , and PCR products were examined by agarose gel electrophoresis and ethidium bromide staining. The expression level of the Act5C transcript in microarray experiments was not significantly altered with FHV infection or replicon expression. Densitometry analysis of PCR products generated from cDNA dilutions that produced submaximal signals showed that FHV infection induced 2.0 ± 0.2 and 2.3 ± 0.3 fold increases in Cct1 and Cct2 mRNA levels, respectively, consistent with quantitative microarray results (see Additional File 1 ). (C) Semi-quantitative RT-PCR validation of Cct1 and Cct2 mRNA upregulation in Drosophila S2 cells expressing an FHV replicon. RT-PCR was performed as described above with total RNA from S2 cells containing the control plasmid pS2F1 fs (upper gel) or FHV replicon-encoding plasmid pS2F1 (lower gel). Densitometry analysis of PCR products as described above showed that FHV replicon expression induced 1.9 ± 0.2 and 2.8 ± 0.9 fold increases in Cct1 and Cct2 mRNA levels, respectively, consistent with quantitative microarray results (see Additional File 2 ).
    Figure Legend Snippet: FHV infection and replicon expression upregulate partially overlapping sets of Drosophila genes . (A) Venn diagram indicating the number of upregulated genes unique to FHV-infected cells (white circle), unique to FHV replicon-expressing cells (grey circle), or upregulated with both (black convergence). Total numbers of genes are given within the indicated regions, and complete lists and descriptions of genes are provided in Table 1 and as Additional Files 1 , 2 and 3 . Specific upregulated genes involved in lipid metabolism, as identified by GO terms, are shown by either their gene symbols or CG designations. (B) Semi-quantitative RT-PCR validation of Cct1 and Cct2 mRNA upregulation in Drosophila S2 cells infected with FHV. Decreasing amounts of cDNA generated by RT with oligo-dT primers and equivalent amounts of total RNA from mock (upper gel) or FHV-infected S2 cells (lower gel) were amplified with gene-specific primers for Drosophila actin ( Act5C) , Cct1 , or Cct2 , and PCR products were examined by agarose gel electrophoresis and ethidium bromide staining. The expression level of the Act5C transcript in microarray experiments was not significantly altered with FHV infection or replicon expression. Densitometry analysis of PCR products generated from cDNA dilutions that produced submaximal signals showed that FHV infection induced 2.0 ± 0.2 and 2.3 ± 0.3 fold increases in Cct1 and Cct2 mRNA levels, respectively, consistent with quantitative microarray results (see Additional File 1 ). (C) Semi-quantitative RT-PCR validation of Cct1 and Cct2 mRNA upregulation in Drosophila S2 cells expressing an FHV replicon. RT-PCR was performed as described above with total RNA from S2 cells containing the control plasmid pS2F1 fs (upper gel) or FHV replicon-encoding plasmid pS2F1 (lower gel). Densitometry analysis of PCR products as described above showed that FHV replicon expression induced 1.9 ± 0.2 and 2.8 ± 0.9 fold increases in Cct1 and Cct2 mRNA levels, respectively, consistent with quantitative microarray results (see Additional File 2 ).

    Techniques Used: Infection, Expressing, Quantitative RT-PCR, Generated, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Microarray, Produced, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

    9) Product Images from "The CCR4–NOT complex maintains liver homeostasis through mRNA deadenylation"

    Article Title: The CCR4–NOT complex maintains liver homeostasis through mRNA deadenylation

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201900494

    Elongated poly(A) tails of RNAs in livers from Cnot1-LKO mice. (A) Poly(A) tail lengths of bulk RNA in livers from Alb-CreER T2 , Cnot1 fl/fl , and Cnot1-LKO mice. The lower graph shows a densitogram of poly(A) tail lengths in each genotype. Signal intensity was normalized to total intensity (%). Values represent the mean of independent experiments ( Alb-CreER T2 ; n = 2, Cnot1 fl/fl and Cnot1-LKO mice; n = 3). (B) Poly(A) tail lengths of the indicated mRNAs in livers from Alb-CreER T2 , Cnot1 fl/fl , and Cnot1-LKO mice. PCR products of RNAs treated with RNase H in the presence of oligo (dT) primer, which indicates that fragments without poly(A) tails were also loaded.
    Figure Legend Snippet: Elongated poly(A) tails of RNAs in livers from Cnot1-LKO mice. (A) Poly(A) tail lengths of bulk RNA in livers from Alb-CreER T2 , Cnot1 fl/fl , and Cnot1-LKO mice. The lower graph shows a densitogram of poly(A) tail lengths in each genotype. Signal intensity was normalized to total intensity (%). Values represent the mean of independent experiments ( Alb-CreER T2 ; n = 2, Cnot1 fl/fl and Cnot1-LKO mice; n = 3). (B) Poly(A) tail lengths of the indicated mRNAs in livers from Alb-CreER T2 , Cnot1 fl/fl , and Cnot1-LKO mice. PCR products of RNAs treated with RNase H in the presence of oligo (dT) primer, which indicates that fragments without poly(A) tails were also loaded.

    Techniques Used: Mouse Assay, Polymerase Chain Reaction

    10) Product Images from "Oligo-Fucoidan prevents IL-6 and CCL2 production and cooperates with p53 to suppress ATM signaling and tumor progression"

    Article Title: Oligo-Fucoidan prevents IL-6 and CCL2 production and cooperates with p53 to suppress ATM signaling and tumor progression

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12111-1

    Oligo-Fucoidan reduces CCL2 and IL-6 expression. CCL2 and IL-6 mRNA expression levels were examined in the p53 +/+ ( a , c , e ) and p53 −/− ( b , d , f ) cells. Cells treated with etoposide (40 μM) alone or etoposide and Oligo-Fucoidan (400 μg/ml) for 24 h were studied ( a , b ). The cells were pre-incubated with or without Oligo-Fucoidan for 6 h before being treated with etoposide for another 18 h ( c , d ). The cells were analyzed after being exposed to etoposide for only 6 h before being treated with Oligo-Fucoidan for another 18 h ( e , f ). The data represent the mean ± SD of three independent experiments. *p
    Figure Legend Snippet: Oligo-Fucoidan reduces CCL2 and IL-6 expression. CCL2 and IL-6 mRNA expression levels were examined in the p53 +/+ ( a , c , e ) and p53 −/− ( b , d , f ) cells. Cells treated with etoposide (40 μM) alone or etoposide and Oligo-Fucoidan (400 μg/ml) for 24 h were studied ( a , b ). The cells were pre-incubated with or without Oligo-Fucoidan for 6 h before being treated with etoposide for another 18 h ( c , d ). The cells were analyzed after being exposed to etoposide for only 6 h before being treated with Oligo-Fucoidan for another 18 h ( e , f ). The data represent the mean ± SD of three independent experiments. *p

    Techniques Used: Expressing, Incubation

    Oligo-Fucoidan and p53 cooperate to regulate DNA damage checkpoints. HCT116 cells were treated with PBS, Oligo-Fucoidan (400 μg/ml) and/or etoposide (40 μM) for 48 h. ( a ) The p53 −/− cell cycle profile was characterized. ( b ) Histograms reveal comparisons of the p53 −/− cell cycle profiles under different experimental settings. ( c ) The p53 +/+ cell cycle profile was assessed. ( d ) Histograms display comparisons of the p53 +/+ cell cycle profiles under different treatment conditions. ( e ) The levels of the indicated apoptotic markers (cleaved PARP and active caspase 3) and their intact molecules were examined. ( f ) The molecules responsible for regulating the G1 and G2/M checkpoints were characterized. The data represent the mean ± SD of three independent experiments. *p
    Figure Legend Snippet: Oligo-Fucoidan and p53 cooperate to regulate DNA damage checkpoints. HCT116 cells were treated with PBS, Oligo-Fucoidan (400 μg/ml) and/or etoposide (40 μM) for 48 h. ( a ) The p53 −/− cell cycle profile was characterized. ( b ) Histograms reveal comparisons of the p53 −/− cell cycle profiles under different experimental settings. ( c ) The p53 +/+ cell cycle profile was assessed. ( d ) Histograms display comparisons of the p53 +/+ cell cycle profiles under different treatment conditions. ( e ) The levels of the indicated apoptotic markers (cleaved PARP and active caspase 3) and their intact molecules were examined. ( f ) The molecules responsible for regulating the G1 and G2/M checkpoints were characterized. The data represent the mean ± SD of three independent experiments. *p

    Techniques Used:

    Oligo-Fucoidan suppresses IL-6 and CCL2 production as well as STAT3 and STAT5 activation. ( a ) HCT116 cells were treated with etoposide (40 μM) alone or etoposide and Oligo-Fucoidan (400 μg/ml) for 24 h. IL-6 and CCL2 secretion was measured after the cells were incubated in treatment-free and serum-free medium for 48 h. The results represent the mean ± SD of three independent experiments. *p
    Figure Legend Snippet: Oligo-Fucoidan suppresses IL-6 and CCL2 production as well as STAT3 and STAT5 activation. ( a ) HCT116 cells were treated with etoposide (40 μM) alone or etoposide and Oligo-Fucoidan (400 μg/ml) for 24 h. IL-6 and CCL2 secretion was measured after the cells were incubated in treatment-free and serum-free medium for 48 h. The results represent the mean ± SD of three independent experiments. *p

    Techniques Used: Activation Assay, Incubation

    Oligo-Fucoidan prevents intrinsic DNA lesions and mitochondrial ROS generation. ( a ) HCT116 cell lines (p53 +/+ and p53 −/− ) were treated with different doses of Oligo-Fucoidan for 48 h. p53, p21 and γ-HAX expression levels were analyzed in the indicated cells. γ-HAX levels were compared between p53 +/+ and p53 −/− cells after Oligo-Fucoidan treatment. ( b ) Mitochondrial superoxide levels were detected by MitoSOX Red, followed by flow cytometry analysis, in cells treated with PBS (MOCK) or Oligo-Fucoidan (400 μg/ml) for 48 h. ( c ) p53, p21 and γ-HAX expression levels were studied in cells exposed to etoposide (40 μM) for different intervals. The protein levels were normalized to those β-actin, and the levels of their corresponding controls were set as 1. ( d ) Mitochondrial superoxide levels were measured after etoposide (40 μM) and Oligo-Fucoidan (400 μg/ml) administration or etoposide treatment alone for 48 h. ( e ) Cell viability was analyzed after the indicated cells being treated with etoposide alone or co-treated with different concentrations of Oligo-Fucoidan for 48 h. The data represent the mean ± SD of three independent experiments. *p
    Figure Legend Snippet: Oligo-Fucoidan prevents intrinsic DNA lesions and mitochondrial ROS generation. ( a ) HCT116 cell lines (p53 +/+ and p53 −/− ) were treated with different doses of Oligo-Fucoidan for 48 h. p53, p21 and γ-HAX expression levels were analyzed in the indicated cells. γ-HAX levels were compared between p53 +/+ and p53 −/− cells after Oligo-Fucoidan treatment. ( b ) Mitochondrial superoxide levels were detected by MitoSOX Red, followed by flow cytometry analysis, in cells treated with PBS (MOCK) or Oligo-Fucoidan (400 μg/ml) for 48 h. ( c ) p53, p21 and γ-HAX expression levels were studied in cells exposed to etoposide (40 μM) for different intervals. The protein levels were normalized to those β-actin, and the levels of their corresponding controls were set as 1. ( d ) Mitochondrial superoxide levels were measured after etoposide (40 μM) and Oligo-Fucoidan (400 μg/ml) administration or etoposide treatment alone for 48 h. ( e ) Cell viability was analyzed after the indicated cells being treated with etoposide alone or co-treated with different concentrations of Oligo-Fucoidan for 48 h. The data represent the mean ± SD of three independent experiments. *p

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    11) Product Images from "Nephrocystin-3 is required for ciliary function in zebrafish embryos"

    Article Title: Nephrocystin-3 is required for ciliary function in zebrafish embryos

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00043.2010

    Disruption of zebrafish nphp3 function during zebrafish embryonic development results in body curvature, hydrocephalus, and pronephric cysts. Sixty hours postfertilization (hpf)-zebrafish embryos injected with 4 ng control-morpholino oligo (MO) has normal morphology ( A ), but injection with 4 ng of nphp3 targeting the translation initiation site of nphp3 mRNA (AUG-MO) led to downward body curvature and hydrocephalus ( D ). Similar phenotypes are seen in embryos injected with 2 ng of splice-blocking (SP-MO; H ). Sections through the hindbrain region, as indicated by white dashed lines in A and D showed the ventricle (star) was of normal size in embryos injected with control MO ( B ) but was enlarged in nphp3 morphant embryo ( E ). Cross sections through the pronephros revealed cysts (asterisks) in nphp3 morphants ( F ), whereas control-MO injected embryos have normal pronephric tubules (arrows; C ). RT-PCR shows that SP-MO altered nphp3 RNA processing ( G ). Two shorter RT-PCR products (stars) were detected solely in SP-MO morphants. *1 indicates the cDNA fragment that skips exon 5, whereas *2 indicates the cDNA fragment that skips exon 5 but includes intron 6. The normal RT-PCR product (arrow) is absent in morphants at 24 hpf. At later stages, the normal product is detected in morphants due to diminishing efficacy of MO knockdown.
    Figure Legend Snippet: Disruption of zebrafish nphp3 function during zebrafish embryonic development results in body curvature, hydrocephalus, and pronephric cysts. Sixty hours postfertilization (hpf)-zebrafish embryos injected with 4 ng control-morpholino oligo (MO) has normal morphology ( A ), but injection with 4 ng of nphp3 targeting the translation initiation site of nphp3 mRNA (AUG-MO) led to downward body curvature and hydrocephalus ( D ). Similar phenotypes are seen in embryos injected with 2 ng of splice-blocking (SP-MO; H ). Sections through the hindbrain region, as indicated by white dashed lines in A and D showed the ventricle (star) was of normal size in embryos injected with control MO ( B ) but was enlarged in nphp3 morphant embryo ( E ). Cross sections through the pronephros revealed cysts (asterisks) in nphp3 morphants ( F ), whereas control-MO injected embryos have normal pronephric tubules (arrows; C ). RT-PCR shows that SP-MO altered nphp3 RNA processing ( G ). Two shorter RT-PCR products (stars) were detected solely in SP-MO morphants. *1 indicates the cDNA fragment that skips exon 5, whereas *2 indicates the cDNA fragment that skips exon 5 but includes intron 6. The normal RT-PCR product (arrow) is absent in morphants at 24 hpf. At later stages, the normal product is detected in morphants due to diminishing efficacy of MO knockdown.

    Techniques Used: Injection, Blocking Assay, Reverse Transcription Polymerase Chain Reaction

    12) Product Images from "The RNA-binding protein ELAV regulates Hox RNA processing, expression and function within the Drosophila nervous system"

    Article Title: The RNA-binding protein ELAV regulates Hox RNA processing, expression and function within the Drosophila nervous system

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.101519

    The RNA-binding protein ELAV regulates Hox RNA processing in the Drosophila CNS. (A,B) The Drosophila Ubx gene produces a spectrum of RNA isoforms via alternative splicing (AS) and alternative polyadenylation (APA). Ubx AS isoforms differ from each other by the presence/absence of small (micro) exons termed M1 and M2; Ubx APA leads to the formation of mRNAs bearing a long or short 3′UTR. PAS1, polyadenylation site 1; PAS2, polyadenylation site 2; 5′E, 5′ exon; 3′E, 3′ exon. 5′ and 3′ mRNA ends are indicated. (C,D) Molecular analysis of Ubx AS profiles in late Drosophila embryos reveals that changes in ELAV expression lead to a significant change in Ubx AS patterns, especially concerning Ubx isoforms Ia and IVa which are over-represented and under-represented, respectively (arrows), in elav mutant ( elav 5 ) embryos. (E) Developmental expression analysis of specific Ubx splicing isoforms in dissected embryonic ventral nerve cords (anterior is to the left) using oligo in situ hybridisation confirms that changes in ELAV expression lead to changes in the expression level of Ubx splicing isoforms in the developing CNS. PS6, parasegment 6. (F,G) Molecular analysis of Ubx APA patterns shows that changes in ELAV level lead to a significant change in the abundance of long and short 3′UTR isoforms at late embryogenesis ( n =3). CDS, coding sequence. Error bars indicate s.e.m. ** P
    Figure Legend Snippet: The RNA-binding protein ELAV regulates Hox RNA processing in the Drosophila CNS. (A,B) The Drosophila Ubx gene produces a spectrum of RNA isoforms via alternative splicing (AS) and alternative polyadenylation (APA). Ubx AS isoforms differ from each other by the presence/absence of small (micro) exons termed M1 and M2; Ubx APA leads to the formation of mRNAs bearing a long or short 3′UTR. PAS1, polyadenylation site 1; PAS2, polyadenylation site 2; 5′E, 5′ exon; 3′E, 3′ exon. 5′ and 3′ mRNA ends are indicated. (C,D) Molecular analysis of Ubx AS profiles in late Drosophila embryos reveals that changes in ELAV expression lead to a significant change in Ubx AS patterns, especially concerning Ubx isoforms Ia and IVa which are over-represented and under-represented, respectively (arrows), in elav mutant ( elav 5 ) embryos. (E) Developmental expression analysis of specific Ubx splicing isoforms in dissected embryonic ventral nerve cords (anterior is to the left) using oligo in situ hybridisation confirms that changes in ELAV expression lead to changes in the expression level of Ubx splicing isoforms in the developing CNS. PS6, parasegment 6. (F,G) Molecular analysis of Ubx APA patterns shows that changes in ELAV level lead to a significant change in the abundance of long and short 3′UTR isoforms at late embryogenesis ( n =3). CDS, coding sequence. Error bars indicate s.e.m. ** P

    Techniques Used: RNA Binding Assay, Expressing, IA, Mutagenesis, In Situ, Hybridization, Sequencing

    13) Product Images from "Transcriptional Activation of the Adenoviral Genome Is Mediated by Capsid Protein VI"

    Article Title: Transcriptional Activation of the Adenoviral Genome Is Mediated by Capsid Protein VI

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002549

    HCMV tegument protein pp71 and HPV minor capsid protein L2 stimulate E1A promoter activation. (A) H1299 cells were transfected with luciferase reporter plasmids coding for E1A promoter and effector plasmids encoding for VI-wt, VI-M1, VI-delta54, HCMV pp71, HPV L2 or an empty vector as negative control. Forty eight hours after transfection, samples were lysed and luciferase activity was measured as described before. Mean and standard deviation are from three independent experiments. (B) H1299 cells were co-transfected with plasmids containing the Ad5 E1-region (pPG-S3) and expression vector for VI-wt, VI-M1, VI-delta54, pp71 or L2. Total-cell extracts were prepared 48 h after transfection and proteins were subjected to IB using Ab against RFP (pVI), pp71 or β-actin as indicated on the right. Note that several splice variants of E1A are recognized depicted by the vertical bar. (C) Cells were transfected as in B and indicated in the legend to C. Forty eight hours after transfection total RNA was prepared from cell lysates and reverse transcribed using oligo-dT primers. E1A mRNA levels were determined using qPCR with E1A specific, exon-spanning primers. Values correspond to the mean of two experiments done in triplicates and the error bar indicates the STD.
    Figure Legend Snippet: HCMV tegument protein pp71 and HPV minor capsid protein L2 stimulate E1A promoter activation. (A) H1299 cells were transfected with luciferase reporter plasmids coding for E1A promoter and effector plasmids encoding for VI-wt, VI-M1, VI-delta54, HCMV pp71, HPV L2 or an empty vector as negative control. Forty eight hours after transfection, samples were lysed and luciferase activity was measured as described before. Mean and standard deviation are from three independent experiments. (B) H1299 cells were co-transfected with plasmids containing the Ad5 E1-region (pPG-S3) and expression vector for VI-wt, VI-M1, VI-delta54, pp71 or L2. Total-cell extracts were prepared 48 h after transfection and proteins were subjected to IB using Ab against RFP (pVI), pp71 or β-actin as indicated on the right. Note that several splice variants of E1A are recognized depicted by the vertical bar. (C) Cells were transfected as in B and indicated in the legend to C. Forty eight hours after transfection total RNA was prepared from cell lysates and reverse transcribed using oligo-dT primers. E1A mRNA levels were determined using qPCR with E1A specific, exon-spanning primers. Values correspond to the mean of two experiments done in triplicates and the error bar indicates the STD.

    Techniques Used: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Negative Control, Activity Assay, Standard Deviation, Expressing, Real-time Polymerase Chain Reaction

    14) Product Images from "Multiple polyadenylation signals and 3′ untranslated sequences are conserved between chicken and human cellular myosin II transcripts"

    Article Title: Multiple polyadenylation signals and 3′ untranslated sequences are conserved between chicken and human cellular myosin II transcripts

    Journal: Gene Expression

    doi:

    Products from PCR amplification of chicken MHC-A cDNA. Chicken MHC-A cDNA was amplified as described in Materials and Methods and analyzed on 2% agarose/TAE gels. Shown above the gel is a schematic of the longest chicken MHC-A mRNA, with the location of the polyadenylation signals indicated. Also indicated is an internal stretch of 15 adenosine residues, including those that end the published sequence (v), and the location of the MHC-A-specific primer used in amplification (black box). The 3′ ends of the messages giving rise to three of the major PCR products are shown next to the corresponding bands on the gel (119, 374, and 458 base pairs). Also shown is a product (83 base pairs) that results from annealing of the oligo-dT adaptor primer to the stretch of 15 adenosine residues located internally (v, A 15 ). M, DNA size markers, sizes (base pairs) indicated to left of gel; PCR, MHC-A PCR products, A n , poly-A tail.
    Figure Legend Snippet: Products from PCR amplification of chicken MHC-A cDNA. Chicken MHC-A cDNA was amplified as described in Materials and Methods and analyzed on 2% agarose/TAE gels. Shown above the gel is a schematic of the longest chicken MHC-A mRNA, with the location of the polyadenylation signals indicated. Also indicated is an internal stretch of 15 adenosine residues, including those that end the published sequence (v), and the location of the MHC-A-specific primer used in amplification (black box). The 3′ ends of the messages giving rise to three of the major PCR products are shown next to the corresponding bands on the gel (119, 374, and 458 base pairs). Also shown is a product (83 base pairs) that results from annealing of the oligo-dT adaptor primer to the stretch of 15 adenosine residues located internally (v, A 15 ). M, DNA size markers, sizes (base pairs) indicated to left of gel; PCR, MHC-A PCR products, A n , poly-A tail.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

    15) Product Images from "The erythropoietin receptor is a downstream effector of Klotho-induced cytoprotection"

    Article Title: The erythropoietin receptor is a downstream effector of Klotho-induced cytoprotection

    Journal: Kidney international

    doi: 10.1038/ki.2013.149

    Expression of EpoR protein and mRNA in rat kidney ( A ) EpoR mRNA expression in the rat kidney or microdissected glomeruli and renal tubules and from normal adult rats at age of 3 months old by RT-PCT. Total RNA was extracted, and complimentary DNA (cDNA) generated with Oligo dT. Specific target genes were examined by PCR with rat specific primers (shown in method section). AQP2: aquaporin-2; CCD: cortical collecting duct; DCT: distal convaluted tubules; Glo: glomeruli; IMCD: inner medullary collecting duct; K: Rat kidney tissue containing cortex and medullar; NaPi-2a: Na-Pi dependent cotransporter-2a; NKCC2: Na-K-2Cl cotransporter; PT: proximal tubules; TAL: thick ascending limb; K-RT: kidney sample with reverse transcriptase omitted; ( B ) EpoR protein expression in BaF3 cell transfected with HA-tagged mouse EpoR or empty vector. Total lysates from native BaF3 and BaF3-HA-EpoR cells were subjected to immunoprecipitation by HA-Resin followed by immunobloting for EpoR with several anti-EpoR antibodies: A82, HA, M-20, Fab 6. Asterisks depict the specific HA-EpoR band. ( C ) Total proteins were extracted from human and rodent kidneys and murine FLC and Ter119-cells, and subjected to immunobloting EpoR with several antibodies against EpoR. A82 is monoclonal rabbit antibody kindly provided by Dr. Steve Elliot; M-20 is polyclonal antibody purchased from Santa Cruz Biotech. Fab 6 is a synthetic human Fab against human EpoR isolated from a phage-displayed library. Asterisks indicate multiple forms of glycosylated EpoR or EpoR fragments. Hu: human; Mu: mouse; FLC: fetal liver cells at E13.5
    Figure Legend Snippet: Expression of EpoR protein and mRNA in rat kidney ( A ) EpoR mRNA expression in the rat kidney or microdissected glomeruli and renal tubules and from normal adult rats at age of 3 months old by RT-PCT. Total RNA was extracted, and complimentary DNA (cDNA) generated with Oligo dT. Specific target genes were examined by PCR with rat specific primers (shown in method section). AQP2: aquaporin-2; CCD: cortical collecting duct; DCT: distal convaluted tubules; Glo: glomeruli; IMCD: inner medullary collecting duct; K: Rat kidney tissue containing cortex and medullar; NaPi-2a: Na-Pi dependent cotransporter-2a; NKCC2: Na-K-2Cl cotransporter; PT: proximal tubules; TAL: thick ascending limb; K-RT: kidney sample with reverse transcriptase omitted; ( B ) EpoR protein expression in BaF3 cell transfected with HA-tagged mouse EpoR or empty vector. Total lysates from native BaF3 and BaF3-HA-EpoR cells were subjected to immunoprecipitation by HA-Resin followed by immunobloting for EpoR with several anti-EpoR antibodies: A82, HA, M-20, Fab 6. Asterisks depict the specific HA-EpoR band. ( C ) Total proteins were extracted from human and rodent kidneys and murine FLC and Ter119-cells, and subjected to immunobloting EpoR with several antibodies against EpoR. A82 is monoclonal rabbit antibody kindly provided by Dr. Steve Elliot; M-20 is polyclonal antibody purchased from Santa Cruz Biotech. Fab 6 is a synthetic human Fab against human EpoR isolated from a phage-displayed library. Asterisks indicate multiple forms of glycosylated EpoR or EpoR fragments. Hu: human; Mu: mouse; FLC: fetal liver cells at E13.5

    Techniques Used: Expressing, Generated, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Isolation

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    Thermo Fisher rna oligos
    HuR interacts with #105708 site in exon 11 of the HTT mRNA. (A) RT-qPCR quantifications of HuR-bound exogenously expressed HTT mRNA 5ʹ fragment (exon 1–10 or exon 1–11) levels in the wild-type mouse striatal cells (STHdh Q7/Q7 ) by <t>RNA-IP</t> (12 technical replicates from 3 biological replicates). IgG was used as a negative control for the IP, and the 18S level was quantified as a baseline control to normalize the signals. HuR interacted with exon 1–11 but not exon 1–10, suggesting that exon 11 contains the HuR-binding site.(B) Similar to (A), but in HD cells (STHdh Q7/Q111 ) transfected with cDNA plasmids expressing EGFP mRNAs containing different candidate binding sites in its 3ʹ UTR region (12 technical replicates from 3 biological replicates). The EGFP mRNA with #105708 site sequences (mouse or human) showed interaction with HuR, suggesting that #105708 (located in exon 11 of HTT mRNA) is a potential HuR-binding site.(C) A representative R-EMSA gel image of different Cy3-labelled RNA <t>oligos</t> of potential binding site sequences after co-incubation with purified HuR-MBP.His proteins (see Fig. S3). The first lanes in each group were loaded by the indicated RNA oligos alone. In the 2 nd and 3 rd lanes in each group, the indicated RNA oligos were incubated and loaded with purified HuR-MBP.His protein at different concentration ratios as indicated. Five repeats were performed, showing consistent results. HuR-binding with the RNA oligo leads to an apparent molecular weight shift to the top (dark arrows), due to mobility block caused by protein-binding.(D) Quantification of the ratio between firefly luciferase and renilla luciferase signals in HD mouse striatal cells (STHdh Q7/Q111 ) transfected with the pmirGLO reporter plasmids (12 technical replicates from 3 biological replicates). The firefly/renilla signal ratio is an indicator of the level of firefly luciferase mRNA, of which the 3ʹ UTR region contained different sequences of potential HuR-binding sites. The HuR knockdown by siRNA caused lowering of the firefly/renilla signal ratio only for #105708-containing plasmids, suggesting that #105708 is likely a functional binding site.(E) RT-qPCR quantifications of HuR-bound endogenous mouse HTT ( msHTT ) mRNA levels in HD mouse striatal cells (STHdh Q7/Q111 ) transfected with pmirGLO plasmids expressing firefly luciferase mRNAs containing different potential binding sites (12 technical replicates from 3 biological replicates). The mRNAs with functional binding sites may compete with endogenous msHTT mRNA for HuR binding (illustrated in the schematic picture in the left panel). Thus, the RNA-IP signals of the corresponding samples could be reduced ( right panel). IgG was used as a negative control for the IP, and the 18S level was quantified as a baseline control to normalize the signals.For all plotted data, error bars represent mean and SEM. The statistical analysis was performed by two-tailed unpaired t tests, ****P
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    HuR interacts with #105708 site in exon 11 of the HTT mRNA. (A) RT-qPCR quantifications of HuR-bound exogenously expressed HTT mRNA 5ʹ fragment (exon 1–10 or exon 1–11) levels in the wild-type mouse striatal cells (STHdh Q7/Q7 ) by RNA-IP (12 technical replicates from 3 biological replicates). IgG was used as a negative control for the IP, and the 18S level was quantified as a baseline control to normalize the signals. HuR interacted with exon 1–11 but not exon 1–10, suggesting that exon 11 contains the HuR-binding site.(B) Similar to (A), but in HD cells (STHdh Q7/Q111 ) transfected with cDNA plasmids expressing EGFP mRNAs containing different candidate binding sites in its 3ʹ UTR region (12 technical replicates from 3 biological replicates). The EGFP mRNA with #105708 site sequences (mouse or human) showed interaction with HuR, suggesting that #105708 (located in exon 11 of HTT mRNA) is a potential HuR-binding site.(C) A representative R-EMSA gel image of different Cy3-labelled RNA oligos of potential binding site sequences after co-incubation with purified HuR-MBP.His proteins (see Fig. S3). The first lanes in each group were loaded by the indicated RNA oligos alone. In the 2 nd and 3 rd lanes in each group, the indicated RNA oligos were incubated and loaded with purified HuR-MBP.His protein at different concentration ratios as indicated. Five repeats were performed, showing consistent results. HuR-binding with the RNA oligo leads to an apparent molecular weight shift to the top (dark arrows), due to mobility block caused by protein-binding.(D) Quantification of the ratio between firefly luciferase and renilla luciferase signals in HD mouse striatal cells (STHdh Q7/Q111 ) transfected with the pmirGLO reporter plasmids (12 technical replicates from 3 biological replicates). The firefly/renilla signal ratio is an indicator of the level of firefly luciferase mRNA, of which the 3ʹ UTR region contained different sequences of potential HuR-binding sites. The HuR knockdown by siRNA caused lowering of the firefly/renilla signal ratio only for #105708-containing plasmids, suggesting that #105708 is likely a functional binding site.(E) RT-qPCR quantifications of HuR-bound endogenous mouse HTT ( msHTT ) mRNA levels in HD mouse striatal cells (STHdh Q7/Q111 ) transfected with pmirGLO plasmids expressing firefly luciferase mRNAs containing different potential binding sites (12 technical replicates from 3 biological replicates). The mRNAs with functional binding sites may compete with endogenous msHTT mRNA for HuR binding (illustrated in the schematic picture in the left panel). Thus, the RNA-IP signals of the corresponding samples could be reduced ( right panel). IgG was used as a negative control for the IP, and the 18S level was quantified as a baseline control to normalize the signals.For all plotted data, error bars represent mean and SEM. The statistical analysis was performed by two-tailed unpaired t tests, ****P

    Journal: RNA Biology

    Article Title: HuR stabilizes HTT mRNA via interacting with its exon 11 in a mutant HTT-dependent manner

    doi: 10.1080/15476286.2020.1712894

    Figure Lengend Snippet: HuR interacts with #105708 site in exon 11 of the HTT mRNA. (A) RT-qPCR quantifications of HuR-bound exogenously expressed HTT mRNA 5ʹ fragment (exon 1–10 or exon 1–11) levels in the wild-type mouse striatal cells (STHdh Q7/Q7 ) by RNA-IP (12 technical replicates from 3 biological replicates). IgG was used as a negative control for the IP, and the 18S level was quantified as a baseline control to normalize the signals. HuR interacted with exon 1–11 but not exon 1–10, suggesting that exon 11 contains the HuR-binding site.(B) Similar to (A), but in HD cells (STHdh Q7/Q111 ) transfected with cDNA plasmids expressing EGFP mRNAs containing different candidate binding sites in its 3ʹ UTR region (12 technical replicates from 3 biological replicates). The EGFP mRNA with #105708 site sequences (mouse or human) showed interaction with HuR, suggesting that #105708 (located in exon 11 of HTT mRNA) is a potential HuR-binding site.(C) A representative R-EMSA gel image of different Cy3-labelled RNA oligos of potential binding site sequences after co-incubation with purified HuR-MBP.His proteins (see Fig. S3). The first lanes in each group were loaded by the indicated RNA oligos alone. In the 2 nd and 3 rd lanes in each group, the indicated RNA oligos were incubated and loaded with purified HuR-MBP.His protein at different concentration ratios as indicated. Five repeats were performed, showing consistent results. HuR-binding with the RNA oligo leads to an apparent molecular weight shift to the top (dark arrows), due to mobility block caused by protein-binding.(D) Quantification of the ratio between firefly luciferase and renilla luciferase signals in HD mouse striatal cells (STHdh Q7/Q111 ) transfected with the pmirGLO reporter plasmids (12 technical replicates from 3 biological replicates). The firefly/renilla signal ratio is an indicator of the level of firefly luciferase mRNA, of which the 3ʹ UTR region contained different sequences of potential HuR-binding sites. The HuR knockdown by siRNA caused lowering of the firefly/renilla signal ratio only for #105708-containing plasmids, suggesting that #105708 is likely a functional binding site.(E) RT-qPCR quantifications of HuR-bound endogenous mouse HTT ( msHTT ) mRNA levels in HD mouse striatal cells (STHdh Q7/Q111 ) transfected with pmirGLO plasmids expressing firefly luciferase mRNAs containing different potential binding sites (12 technical replicates from 3 biological replicates). The mRNAs with functional binding sites may compete with endogenous msHTT mRNA for HuR binding (illustrated in the schematic picture in the left panel). Thus, the RNA-IP signals of the corresponding samples could be reduced ( right panel). IgG was used as a negative control for the IP, and the 18S level was quantified as a baseline control to normalize the signals.For all plotted data, error bars represent mean and SEM. The statistical analysis was performed by two-tailed unpaired t tests, ****P

    Article Snippet: Then the titrated HuR protein (2 mg/mL) and RNA oligos were dissolved in the EMSA interaction buffer (1 × R-EMSA Binding Buffer (Thermo Fisher Scientific, #20158) + 5% glycerol + 100 μg/mL tRNA+1× RNase inhibitor (Thermo Fisher Scientific, #N8080119)), and then incubated for 30 min at room temperature (25 °C).

    Techniques: Quantitative RT-PCR, Negative Control, Binding Assay, Transfection, Expressing, Incubation, Purification, Concentration Assay, Molecular Weight, Blocking Assay, Protein Binding, Luciferase, Functional Assay, Two Tailed Test

    Ab-oligo conjugate staining validation. The staining pattern of each Ab-oligo conjugate was validated by staining serial sections using conventional indirect IF ( 1 ° + 2 ° ), the Ab-oligo conjugate ( 1 ° ) detected with a conventional fluorophore-labeled secondary antibody ( 2 ° ), the Ab-oligo conjugate detected using the complementary IS ( Ab - oligo + IS ), and direct IF using fluorophore (FL) conjugated 1 ° antibody. The appropriate negative control image is located below their corresponding antibody stained image. Ab-oligo staining pattern was verified for (a) CK5, (b) CK8, (c) CK19, (d) PCNA, (e) Ki67, (f) E-Cad, (g) HER2, (h) α -SMA, (i) CD44, (j) CoxIV, (k) CD4, and (l) CD3. SBR was calculated for (m) each indirect IF and Ab - oligo + 2 ° image and compared with (n) SBR calculated for each fluorophore (FL) conjugated 1 ° antibody and Ab - oligo + IS image. The 40 - μ m scale bars are displayed in all images.

    Journal: Journal of Biomedical Optics

    Article Title: Oligonucleotide conjugated antibodies permit highly multiplexed immunofluorescence for future use in clinical histopathology

    doi: 10.1117/1.JBO.25.5.056004

    Figure Lengend Snippet: Ab-oligo conjugate staining validation. The staining pattern of each Ab-oligo conjugate was validated by staining serial sections using conventional indirect IF ( 1 ° + 2 ° ), the Ab-oligo conjugate ( 1 ° ) detected with a conventional fluorophore-labeled secondary antibody ( 2 ° ), the Ab-oligo conjugate detected using the complementary IS ( Ab - oligo + IS ), and direct IF using fluorophore (FL) conjugated 1 ° antibody. The appropriate negative control image is located below their corresponding antibody stained image. Ab-oligo staining pattern was verified for (a) CK5, (b) CK8, (c) CK19, (d) PCNA, (e) Ki67, (f) E-Cad, (g) HER2, (h) α -SMA, (i) CD44, (j) CoxIV, (k) CD4, and (l) CD3. SBR was calculated for (m) each indirect IF and Ab - oligo + 2 ° image and compared with (n) SBR calculated for each fluorophore (FL) conjugated 1 ° antibody and Ab - oligo + IS image. The 40 - μ m scale bars are displayed in all images.

    Article Snippet: After 5 min in RT diH 2 O, the slides were washed in PBS, pH 7.4 at RT for an additional 5 min. Antibody staining studies were completed using four groups for staining pattern validation, including (1) conventional indirect IF with unconjugated primary and fluorophore conjugated secondary antibody, (2) the Ab-oligo conjugate plus the same fluorophore conjugated secondary antibody, (3) the Ab-oligo conjugate plus the complementary fluorophore conjugated IS, and (4) the fluorophore labeled primary.

    Techniques: Staining, Labeling, Negative Control

    Induction of SASP by activating the cytoplasmic DNA sensing pathway. a Senescent TIG-3 cells induced by oncogenic Ras expression (lane 2–4) were transfected with previously validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ), RT-qPCR analysis of SASP factor gene expression ( b ), analysis of intracellular ROS levels ( c ) or immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 40,6-diamidino-2-phenylindole [blue]) on day 4 ( d ). The representative data from three independent experiments are shown. Tubulin was used as a loading control ( a ). For all graphs, error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (* P

    Journal: Nature Communications

    Article Title: Downregulation of cytoplasmic DNases is implicated in cytoplasmic DNA accumulation and SASP in senescent cells

    doi: 10.1038/s41467-018-03555-8

    Figure Lengend Snippet: Induction of SASP by activating the cytoplasmic DNA sensing pathway. a Senescent TIG-3 cells induced by oncogenic Ras expression (lane 2–4) were transfected with previously validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ), RT-qPCR analysis of SASP factor gene expression ( b ), analysis of intracellular ROS levels ( c ) or immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 40,6-diamidino-2-phenylindole [blue]) on day 4 ( d ). The representative data from three independent experiments are shown. Tubulin was used as a loading control ( a ). For all graphs, error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (* P

    Article Snippet: RNAi was performed by the transfection of siRNA oligos using the Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining, Standard Deviation

    The knockdown of cytoplasmic DNases activates the IFN-β pathway. a – c Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right ( a ), isolation of cytoplasmic fraction followed by qPCR analysis of chromosomal DNA ( b ) or qPCR analysis of SASP factor gene expression ( c ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Downregulation of cytoplasmic DNases is implicated in cytoplasmic DNA accumulation and SASP in senescent cells

    doi: 10.1038/s41467-018-03555-8

    Figure Lengend Snippet: The knockdown of cytoplasmic DNases activates the IFN-β pathway. a – c Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right ( a ), isolation of cytoplasmic fraction followed by qPCR analysis of chromosomal DNA ( b ) or qPCR analysis of SASP factor gene expression ( c ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (** P

    Article Snippet: RNAi was performed by the transfection of siRNA oligos using the Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions.

    Techniques: Transfection, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

    GGTase3 geranylgeranylates FBXL2 and is required for its localization to cellular membranes. (a) Recombinant GGtase3 geranylgeranylates purified FBXL2. Indicated amounts of purified FBXL2 were incubated with 100 ng of purified GGTase3 (either tagged [T] or untagged [UT] versions) to carry out in vitro geranylgeranylation assay using saturating concentrations of tritiated [H 3 ]-GGPP as described in methods. Each data point represents mean+/− SD of three biological replicates. Michaelis-Menten kinetics was used to generate an apparent K m value of 1.2μM using Prism Graphpad software. (b) In vitro geranylgeranylation assay was carried out and measured as in (a) using 10 μM of purified FBXL2, FBXW7, or K-RAS4B and 100 ng of purified GGTase3. Bar graphs represent mean +/− SD from three biological replicates. Source data for panels a and b are available with the paper online. (c) RPE1-HTERT cells were cotransfected with the indicated plasmids and processed for the detection of geranylgeranylated FBXL2 using a “Click-IT” assay, as described in methods. The experiment was repeated three times. Representative result is shown. Uncropped blot/gel images are shown in Supplementary Data Set 1 . (d) HeLa cells were transfected with the indicated siRNA oligos and cDNAs. Twenty-four hours post-transfection cells were incubated with geranylgeranyl-azide for 16 hours, harvested, lysed, and azide selective ligation reaction with sDIBO-Biotin was performed for one hour to label geranylgeranylated proteins via copper-free “Click-IT” reaction. After immunoprecipitation with an anti-HA antibody, immunoblots were carried out. The experiment was repeated four times. Representative result is shown. Uncropped blot/gel images are shown in Supplementary Data Set 1 . (e) HeLa cells were transfected first with the indicated siRNA oligos and then with the indicated GFP-tagged proteins. Live cell confocal imaging was carried out as described in methods. Images show representative frames of three independent experiments. Bar size: 10 μm.

    Journal: Nature structural & molecular biology

    Article Title: GGTase3 is a Newly Identified Geranylgeranyltransferase Targeting a Ubiquitin Ligase

    doi: 10.1038/s41594-019-0249-3

    Figure Lengend Snippet: GGTase3 geranylgeranylates FBXL2 and is required for its localization to cellular membranes. (a) Recombinant GGtase3 geranylgeranylates purified FBXL2. Indicated amounts of purified FBXL2 were incubated with 100 ng of purified GGTase3 (either tagged [T] or untagged [UT] versions) to carry out in vitro geranylgeranylation assay using saturating concentrations of tritiated [H 3 ]-GGPP as described in methods. Each data point represents mean+/− SD of three biological replicates. Michaelis-Menten kinetics was used to generate an apparent K m value of 1.2μM using Prism Graphpad software. (b) In vitro geranylgeranylation assay was carried out and measured as in (a) using 10 μM of purified FBXL2, FBXW7, or K-RAS4B and 100 ng of purified GGTase3. Bar graphs represent mean +/− SD from three biological replicates. Source data for panels a and b are available with the paper online. (c) RPE1-HTERT cells were cotransfected with the indicated plasmids and processed for the detection of geranylgeranylated FBXL2 using a “Click-IT” assay, as described in methods. The experiment was repeated three times. Representative result is shown. Uncropped blot/gel images are shown in Supplementary Data Set 1 . (d) HeLa cells were transfected with the indicated siRNA oligos and cDNAs. Twenty-four hours post-transfection cells were incubated with geranylgeranyl-azide for 16 hours, harvested, lysed, and azide selective ligation reaction with sDIBO-Biotin was performed for one hour to label geranylgeranylated proteins via copper-free “Click-IT” reaction. After immunoprecipitation with an anti-HA antibody, immunoblots were carried out. The experiment was repeated four times. Representative result is shown. Uncropped blot/gel images are shown in Supplementary Data Set 1 . (e) HeLa cells were transfected first with the indicated siRNA oligos and then with the indicated GFP-tagged proteins. Live cell confocal imaging was carried out as described in methods. Images show representative frames of three independent experiments. Bar size: 10 μm.

    Article Snippet: Cells were either with siRNA oligos and/or plasmids for the times indicated in figure legends using Lipofactemine®3000 reagent for 1 hour at 37°C prior to metabolic labeling with geranylgeranly azide (30uM, Thermo Scientific # C10249) for 24 hours in DMEM medium supplemented with 10% dialyzed FBS and 5 mM sodium pyruvate.

    Techniques: Recombinant, Purification, Incubation, In Vitro, Software, Transfection, Ligation, Immunoprecipitation, Western Blot, Imaging