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Promega protector rnasin inhibitor
Protector Rnasin Inhibitor, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 79 stars, based on 1 article reviews
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protector rnasin inhibitor - by Bioz Stars, 2020-03
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Polymerase Chain Reaction:

Article Title: Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl
Article Snippet: Paragraph title: Reverse transcription and analysis of gene expression by PCR ... Two micrograms of total RNA were reverse-transcribed to cDNA using an oligo-(dT) of 17 bp, 30 units of the AMV reverse-transcriptase (Promega) and 40 units of protector RNasin inhibitor (Promega).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl
Article Snippet: Two micrograms of total RNA were reverse-transcribed to cDNA using an oligo-(dT) of 17 bp, 30 units of the AMV reverse-transcriptase (Promega) and 40 units of protector RNasin inhibitor (Promega). .. Semi-quantitative RT-PCR was performed in a final volume of 20 µL with 1·2 µL of the diluted cDNA, 0·5 units of GoTaq Polymerase (Promega), 5 m m dNTPs, and 10 pmol of each gene-specific primer (Table ).

RNA Extraction:

Article Title: Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl
Article Snippet: Total RNA was prepared using an RNA extraction kit (Nucleospin RNA plant, Macherey-Nagel, Düren, Germany) and DNase I (Promega, Madison, WI, USA) from 200–400 mg of hypocotyls. .. Two micrograms of total RNA were reverse-transcribed to cDNA using an oligo-(dT) of 17 bp, 30 units of the AMV reverse-transcriptase (Promega) and 40 units of protector RNasin inhibitor (Promega).

Expressing:

Article Title: Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl
Article Snippet: Paragraph title: Reverse transcription and analysis of gene expression by PCR ... Two micrograms of total RNA were reverse-transcribed to cDNA using an oligo-(dT) of 17 bp, 30 units of the AMV reverse-transcriptase (Promega) and 40 units of protector RNasin inhibitor (Promega).

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  • 99
    Promega tnf α
    Fig. 5. Inhibition of synthesis of the luciferase reporter gene by P56 in vivo . ( A ) Interaction of P48/Int-6 with P56 but not MP56. pCMV-P56 (lanes 1 and 3) or pCMV-MP56 (lanes 2 and 4) was co-transfected with pCMV-P48Fl into cells. At 48 h post-transfection, cells were harvested and whole-cell extracts were prepared. A 50 µg aliquot of total cell protein was subjected to gel electrophoresis followed by western blotting with P56 antibody (lanes 1 and 2). A 1 mg aliquot of cell protein was subjected to immunoprecipitation with anti-Flag-conjugated Sepharose beads followed by western blot analysis with P56 antibody (lanes 3 and 4). ( B ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (bar 4), pCMV-MP56 (bar 5), pCMV-DRBP76 (bar 3) or the empty expression vector (bars 1 and 2). After 48 h, cells were treated with <t>TNF-α</t> (bars 2–5) for 4 h. Cell extracts were made and luciferase activity was measured. The averages of results from three experiments are shown. ( C ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (+) or vector (–). At 48 h post-transfection, cells were treated with TNF-α for 4 h. Cells were harvested and total RNA was isolated for RNase protection assay. A 40 µg aliquot of total RNA was hybridized with 32 P-labeled Luc (370 bases) and γ-actin (140 bases) antisense RNA probes shown on the left as undigested probes. Following RNase digestion, the protected RNA probes were resolved in a 6% polyacrylamide, 8 M urea gel. Luciferase mRNA levels, shown on the right as protected probes, were quantified by phosphorimager and, after normalizing against the γ-actin mRNA levels, they were comparable in the two samples. ( D ) Cells were co-transfected with E-selectin-Luc and vector, pCMV-P56 or pCMV-MP56, as indicated. The experimental protocol was the same as in (B). ( E ) The same three cell extracts from (D) were western blotted with P56 antibody.
    Tnf α, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tnf α/product/Promega
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    85
    Promega sg220
    Liver uptake of <t>LNP-SG220</t> in CD1 mice. ( a ) Denaturing 10% PAGE analysis of RNase protection assay to monitor SG220 present in livers of CD1 mice at the indicated doses and time points. A mouse injected with PBS only was used as a control (PBS). Bands corresponding to undigested probe and probe protected after RNase A/T1 digestion are labeled. For quantification, the amounts of SG220 indicated in the last four lanes were spiked into 50 µg of total liver RNA from untreated mice to generate a calibration curve for the quantification of the liver uptake of SG220. PP, protected probe; UP, undigested full-length probe. ( b ) Plots of background corrected counts ( 32 P) for individual mice for dosing groups 0.5 and 2.5 mg/kg. LNP, lipid nanoparticles.
    Sg220, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Promega rnase h protection assay
    U1C depletion results in specific alternative splicing alterations in HeLa cells: Specificity and validation. ( A ) U1C knockdown (kd) in HeLa cells. Whole cell lysates were analyzed by SDS-PAGE and Western blot detecting U1C and γ-tubulin. U1 snRNA steady-state levels were analyzed by Northern blotting with probes specific for U1 snRNA and, as a loading control, U3 snoRNA. HeLa cells after U1C knockdown (ΔC) and luciferase-siRNA treated control cells (ctr) were compared. ( B ) Graphical overview of U1C-dependent alternative splicing targets identified by RNA-Seq analysis. ( C ) U1 snRNA blocking in HeLa cells. The efficiency of U1 snRNA blocking was determined by <t>RNase</t> H protection and silver staining. The positions of the full-length U1 snRNA (U1 uncut), the RNase H-cleaved U1 snRNA (U1 cut), and the U2 snRNA (as a control) are marked on the right. ( D ) Alternative splicing patterns of selected U1C target genes (names above the lanes) were analyzed by RT-PCR, using total RNA from HeLa cells after U1C-knockdown (ctr vs. ΔC) or U1 snRNA blocking (ctr vs. U1). Target-specific primers (arrows in the schematics on the right of the panels) were designed to amplify both alternative splicing isoforms. M , DNA size markers (in bp). Upper panel: Top and lower bands represent exon inclusion and skipping products, respectively; an unspecific product for SNHG5 is marked by open circles between the lanes. Lower panel: For MARCH7 top and lower bands reflect usage of the proximal and distal 5′ splice site, respectively. For UFM1 three alternative 5′ splice sites are activated upon U1C knockdown labeled with 1, 2, and 3 on the right.
    Rnase H Protection Assay, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Promega mouse monoclonal anti β galactosidase
    Acetylation of GATA-1 causes ubiquitination. ( A ) and <t>β-galactosidase</t> with (+) or without (−) TSA treatment. Lower panel: Western blot showing the levels of ubiquitinated GATA-1 (Ub-GATA-1), acetylated GATA-1 (Ac-GATA-1) and total GATA-1 following immunoprecipitation from transfected Cos 7 cells treated with MG-132 with (+) or without (−) TSA. ( B ) p300 but not p300dHAT decreases GATA-1 levels. NIH3T3 cells were transfected with expression vectors for GATA-1 and p300 or its acetyltransferase mutant, p300dHAT. Whole-cell extracts were used in a Western blot with anti-GATA-1 antibody. β-Galactosidase is the cotransfection control. ( C ). Total GATA-1 levels decrease more in Cos 7 cells (A, upper panel) than BM-SCF cells (C, second panel) upon TSA treatment. GATA-1 regulates its own promoter in haemopoietic cells but is controlled by the EF1α promoter in Cos 7 cells. To prevent TSA increasing GATA-1 levels in BM-SCF cells via GATA-1 autoregulation, we added the transcription inhibitor α-amanitin (α-aman). A more substantial decrease is now seen (fourth panel). An anti-β-tubulin Western confirms that equivalent cell numbers were used (lower panel). ( D ) resulted in a similar increase in GATA-1 stability, suggesting that it is unlikely that loss of ubiquitination sites caused the increased stability. ( E ).
    Mouse Monoclonal Anti β Galactosidase, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fig. 5. Inhibition of synthesis of the luciferase reporter gene by P56 in vivo . ( A ) Interaction of P48/Int-6 with P56 but not MP56. pCMV-P56 (lanes 1 and 3) or pCMV-MP56 (lanes 2 and 4) was co-transfected with pCMV-P48Fl into cells. At 48 h post-transfection, cells were harvested and whole-cell extracts were prepared. A 50 µg aliquot of total cell protein was subjected to gel electrophoresis followed by western blotting with P56 antibody (lanes 1 and 2). A 1 mg aliquot of cell protein was subjected to immunoprecipitation with anti-Flag-conjugated Sepharose beads followed by western blot analysis with P56 antibody (lanes 3 and 4). ( B ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (bar 4), pCMV-MP56 (bar 5), pCMV-DRBP76 (bar 3) or the empty expression vector (bars 1 and 2). After 48 h, cells were treated with TNF-α (bars 2–5) for 4 h. Cell extracts were made and luciferase activity was measured. The averages of results from three experiments are shown. ( C ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (+) or vector (–). At 48 h post-transfection, cells were treated with TNF-α for 4 h. Cells were harvested and total RNA was isolated for RNase protection assay. A 40 µg aliquot of total RNA was hybridized with 32 P-labeled Luc (370 bases) and γ-actin (140 bases) antisense RNA probes shown on the left as undigested probes. Following RNase digestion, the protected RNA probes were resolved in a 6% polyacrylamide, 8 M urea gel. Luciferase mRNA levels, shown on the right as protected probes, were quantified by phosphorimager and, after normalizing against the γ-actin mRNA levels, they were comparable in the two samples. ( D ) Cells were co-transfected with E-selectin-Luc and vector, pCMV-P56 or pCMV-MP56, as indicated. The experimental protocol was the same as in (B). ( E ) The same three cell extracts from (D) were western blotted with P56 antibody.

    Journal: The EMBO Journal

    Article Title: A new pathway of translational regulation mediated by eukaryotic initiation factor 3

    doi: 10.1093/emboj/19.24.6891

    Figure Lengend Snippet: Fig. 5. Inhibition of synthesis of the luciferase reporter gene by P56 in vivo . ( A ) Interaction of P48/Int-6 with P56 but not MP56. pCMV-P56 (lanes 1 and 3) or pCMV-MP56 (lanes 2 and 4) was co-transfected with pCMV-P48Fl into cells. At 48 h post-transfection, cells were harvested and whole-cell extracts were prepared. A 50 µg aliquot of total cell protein was subjected to gel electrophoresis followed by western blotting with P56 antibody (lanes 1 and 2). A 1 mg aliquot of cell protein was subjected to immunoprecipitation with anti-Flag-conjugated Sepharose beads followed by western blot analysis with P56 antibody (lanes 3 and 4). ( B ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (bar 4), pCMV-MP56 (bar 5), pCMV-DRBP76 (bar 3) or the empty expression vector (bars 1 and 2). After 48 h, cells were treated with TNF-α (bars 2–5) for 4 h. Cell extracts were made and luciferase activity was measured. The averages of results from three experiments are shown. ( C ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (+) or vector (–). At 48 h post-transfection, cells were treated with TNF-α for 4 h. Cells were harvested and total RNA was isolated for RNase protection assay. A 40 µg aliquot of total RNA was hybridized with 32 P-labeled Luc (370 bases) and γ-actin (140 bases) antisense RNA probes shown on the left as undigested probes. Following RNase digestion, the protected RNA probes were resolved in a 6% polyacrylamide, 8 M urea gel. Luciferase mRNA levels, shown on the right as protected probes, were quantified by phosphorimager and, after normalizing against the γ-actin mRNA levels, they were comparable in the two samples. ( D ) Cells were co-transfected with E-selectin-Luc and vector, pCMV-P56 or pCMV-MP56, as indicated. The experimental protocol was the same as in (B). ( E ) The same three cell extracts from (D) were western blotted with P56 antibody.

    Article Snippet: After 48 h, cells were induced with 20 ng/ml TNF-α for 4 h. Cell extracts were prepared in 1× reporter lysis buffer (Promega) and luciferase activity was measured using the luciferase reporter gene assay kit (Promega).

    Techniques: Inhibition, Luciferase, In Vivo, Transfection, Nucleic Acid Electrophoresis, Western Blot, Immunoprecipitation, Expressing, Plasmid Preparation, Activity Assay, Isolation, Rnase Protection Assay, Labeling

    Liver uptake of LNP-SG220 in CD1 mice. ( a ) Denaturing 10% PAGE analysis of RNase protection assay to monitor SG220 present in livers of CD1 mice at the indicated doses and time points. A mouse injected with PBS only was used as a control (PBS). Bands corresponding to undigested probe and probe protected after RNase A/T1 digestion are labeled. For quantification, the amounts of SG220 indicated in the last four lanes were spiked into 50 µg of total liver RNA from untreated mice to generate a calibration curve for the quantification of the liver uptake of SG220. PP, protected probe; UP, undigested full-length probe. ( b ) Plots of background corrected counts ( 32 P) for individual mice for dosing groups 0.5 and 2.5 mg/kg. LNP, lipid nanoparticles.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Minimal-length Synthetic shRNAs Formulated with Lipid Nanoparticles are Potent Inhibitors of Hepatitis C Virus IRES-linked Gene Expression in Mice

    doi: 10.1038/mtna.2013.50

    Figure Lengend Snippet: Liver uptake of LNP-SG220 in CD1 mice. ( a ) Denaturing 10% PAGE analysis of RNase protection assay to monitor SG220 present in livers of CD1 mice at the indicated doses and time points. A mouse injected with PBS only was used as a control (PBS). Bands corresponding to undigested probe and probe protected after RNase A/T1 digestion are labeled. For quantification, the amounts of SG220 indicated in the last four lanes were spiked into 50 µg of total liver RNA from untreated mice to generate a calibration curve for the quantification of the liver uptake of SG220. PP, protected probe; UP, undigested full-length probe. ( b ) Plots of background corrected counts ( 32 P) for individual mice for dosing groups 0.5 and 2.5 mg/kg. LNP, lipid nanoparticles.

    Article Snippet: The 32 P-labeled probe specific for the detection of SG220 was prepared by in vitro transcription using T7 RNA polymerase (Promega) in the presence of [α32 P]CTP (Perkin-Elmer, Boston, MA).

    Techniques: Mouse Assay, Polyacrylamide Gel Electrophoresis, Rnase Protection Assay, Injection, Labeling

    Kinetics of uptake of sshRNA and lipid components of LNP-formulated SG220 into mouse liver. CD1 ICR mice were administered one intravenous injection of LNP-formulated SG220 at 2.5 mg/kg ( n = 4 for each time point). Levels of sshRNA and CHE in the serum and liver at various times after dosing were quantified as described in “Materials and Methods.” Liver uptake is presented as a percentage of the initial dose. The error bars show mean ± SEM from four mice. LNP, lipid nanoparticles.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Minimal-length Synthetic shRNAs Formulated with Lipid Nanoparticles are Potent Inhibitors of Hepatitis C Virus IRES-linked Gene Expression in Mice

    doi: 10.1038/mtna.2013.50

    Figure Lengend Snippet: Kinetics of uptake of sshRNA and lipid components of LNP-formulated SG220 into mouse liver. CD1 ICR mice were administered one intravenous injection of LNP-formulated SG220 at 2.5 mg/kg ( n = 4 for each time point). Levels of sshRNA and CHE in the serum and liver at various times after dosing were quantified as described in “Materials and Methods.” Liver uptake is presented as a percentage of the initial dose. The error bars show mean ± SEM from four mice. LNP, lipid nanoparticles.

    Article Snippet: The 32 P-labeled probe specific for the detection of SG220 was prepared by in vitro transcription using T7 RNA polymerase (Promega) in the presence of [α32 P]CTP (Perkin-Elmer, Boston, MA).

    Techniques: Mouse Assay, Injection

    Inhibition of HCV IRES-dependent luciferase gene expression in mouse liver by HCV sshRNA SG220. Seven days after the mice were hydrodynamically injected with the HCV-IRES-fLucplasmid pSG231, either SG220 or an irrelevant (sequence-scrambled) sshRNA (irr sshRNA) formulated with lipid nanoparticles at the indicated doses, or PBS, was injected intravenously at low pressure. At the indicated time points post-sshRNA injection, luciferin was injected intraperitoneally and luciferase gene expression in the mouse liver was detected by in vivo bioluminescence imaging. The luciferase gene expression at the indicated time points is presented as a percentage of that prior to the sshRNA injection.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Minimal-length Synthetic shRNAs Formulated with Lipid Nanoparticles are Potent Inhibitors of Hepatitis C Virus IRES-linked Gene Expression in Mice

    doi: 10.1038/mtna.2013.50

    Figure Lengend Snippet: Inhibition of HCV IRES-dependent luciferase gene expression in mouse liver by HCV sshRNA SG220. Seven days after the mice were hydrodynamically injected with the HCV-IRES-fLucplasmid pSG231, either SG220 or an irrelevant (sequence-scrambled) sshRNA (irr sshRNA) formulated with lipid nanoparticles at the indicated doses, or PBS, was injected intravenously at low pressure. At the indicated time points post-sshRNA injection, luciferin was injected intraperitoneally and luciferase gene expression in the mouse liver was detected by in vivo bioluminescence imaging. The luciferase gene expression at the indicated time points is presented as a percentage of that prior to the sshRNA injection.

    Article Snippet: The 32 P-labeled probe specific for the detection of SG220 was prepared by in vitro transcription using T7 RNA polymerase (Promega) in the presence of [α32 P]CTP (Perkin-Elmer, Boston, MA).

    Techniques: Inhibition, Luciferase, Expressing, Mouse Assay, Injection, Sequencing, In Vivo, Imaging

    Gene silencing activity of HCV sshRNAs in vitro . ( a ) Secondary structure of the HCV IRES with the region targeted by sshRNA SG220 indicated by the bracket. ( b ) Potency of SG220 in suppressing luciferase gene expression driven by the HCV-IRES in 293FT cells. SG220 (filled diamonds); SG221C (scrambled control) (squares). The results shown are the mean va lues ± SEM from triplicate transfections.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Minimal-length Synthetic shRNAs Formulated with Lipid Nanoparticles are Potent Inhibitors of Hepatitis C Virus IRES-linked Gene Expression in Mice

    doi: 10.1038/mtna.2013.50

    Figure Lengend Snippet: Gene silencing activity of HCV sshRNAs in vitro . ( a ) Secondary structure of the HCV IRES with the region targeted by sshRNA SG220 indicated by the bracket. ( b ) Potency of SG220 in suppressing luciferase gene expression driven by the HCV-IRES in 293FT cells. SG220 (filled diamonds); SG221C (scrambled control) (squares). The results shown are the mean va lues ± SEM from triplicate transfections.

    Article Snippet: The 32 P-labeled probe specific for the detection of SG220 was prepared by in vitro transcription using T7 RNA polymerase (Promega) in the presence of [α32 P]CTP (Perkin-Elmer, Boston, MA).

    Techniques: Activity Assay, In Vitro, Luciferase, Expressing, Transfection

    Lack of immune stimulatory effect of SG220 in vitro and in vivo . ( a ) In vitro analysis. Twenty nmol/l sshRNAs SG220 or SG273 were transfected into human MRC-5 cells in triplicate. Untransfected (N/A) cells and cells transfected with Lipofectamine 2000 alone (Lipo2K) served as negative controls. The numbers shown are the mean and standard deviations of the mean of the indicated cytokine mRNAs relative to the untransfected control and normalized to GAPDH. ( b, c ) In vivo analysis. CD1 ICR mice were administered 2.5 mg/kg LNP-formulated sshRNA (SG220) or LNP-formulated control siRNAs (LUC-U/U, LUC) by intravenous injection. At the designated time point, blood was collected by cardiac puncture and processed as plasma for cytokine determination, and liver was excised and placed in RNAlater (Sigma–Aldrich) for IFIT1 mRNA analysis. The values shown are mean and standard deviation of measurements from each group of four mice. ( b ) Plasma cytokines (left) and liver IFIT mRNA (right), determined 4 hours after administration. ( c ) Time course of SG220-mediated plasma cytokine (left) and liver IFIT1 mRNA (right) induction. LNP, lipid nanoparticles.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Minimal-length Synthetic shRNAs Formulated with Lipid Nanoparticles are Potent Inhibitors of Hepatitis C Virus IRES-linked Gene Expression in Mice

    doi: 10.1038/mtna.2013.50

    Figure Lengend Snippet: Lack of immune stimulatory effect of SG220 in vitro and in vivo . ( a ) In vitro analysis. Twenty nmol/l sshRNAs SG220 or SG273 were transfected into human MRC-5 cells in triplicate. Untransfected (N/A) cells and cells transfected with Lipofectamine 2000 alone (Lipo2K) served as negative controls. The numbers shown are the mean and standard deviations of the mean of the indicated cytokine mRNAs relative to the untransfected control and normalized to GAPDH. ( b, c ) In vivo analysis. CD1 ICR mice were administered 2.5 mg/kg LNP-formulated sshRNA (SG220) or LNP-formulated control siRNAs (LUC-U/U, LUC) by intravenous injection. At the designated time point, blood was collected by cardiac puncture and processed as plasma for cytokine determination, and liver was excised and placed in RNAlater (Sigma–Aldrich) for IFIT1 mRNA analysis. The values shown are mean and standard deviation of measurements from each group of four mice. ( b ) Plasma cytokines (left) and liver IFIT mRNA (right), determined 4 hours after administration. ( c ) Time course of SG220-mediated plasma cytokine (left) and liver IFIT1 mRNA (right) induction. LNP, lipid nanoparticles.

    Article Snippet: The 32 P-labeled probe specific for the detection of SG220 was prepared by in vitro transcription using T7 RNA polymerase (Promega) in the presence of [α32 P]CTP (Perkin-Elmer, Boston, MA).

    Techniques: In Vitro, In Vivo, Transfection, Mouse Assay, Injection, Standard Deviation

    U1C depletion results in specific alternative splicing alterations in HeLa cells: Specificity and validation. ( A ) U1C knockdown (kd) in HeLa cells. Whole cell lysates were analyzed by SDS-PAGE and Western blot detecting U1C and γ-tubulin. U1 snRNA steady-state levels were analyzed by Northern blotting with probes specific for U1 snRNA and, as a loading control, U3 snoRNA. HeLa cells after U1C knockdown (ΔC) and luciferase-siRNA treated control cells (ctr) were compared. ( B ) Graphical overview of U1C-dependent alternative splicing targets identified by RNA-Seq analysis. ( C ) U1 snRNA blocking in HeLa cells. The efficiency of U1 snRNA blocking was determined by RNase H protection and silver staining. The positions of the full-length U1 snRNA (U1 uncut), the RNase H-cleaved U1 snRNA (U1 cut), and the U2 snRNA (as a control) are marked on the right. ( D ) Alternative splicing patterns of selected U1C target genes (names above the lanes) were analyzed by RT-PCR, using total RNA from HeLa cells after U1C-knockdown (ctr vs. ΔC) or U1 snRNA blocking (ctr vs. U1). Target-specific primers (arrows in the schematics on the right of the panels) were designed to amplify both alternative splicing isoforms. M , DNA size markers (in bp). Upper panel: Top and lower bands represent exon inclusion and skipping products, respectively; an unspecific product for SNHG5 is marked by open circles between the lanes. Lower panel: For MARCH7 top and lower bands reflect usage of the proximal and distal 5′ splice site, respectively. For UFM1 three alternative 5′ splice sites are activated upon U1C knockdown labeled with 1, 2, and 3 on the right.

    Journal: PLoS Genetics

    Article Title: A Novel Intra-U1 snRNP Cross-Regulation Mechanism: Alternative Splicing Switch Links U1C and U1-70K Expression

    doi: 10.1371/journal.pgen.1003856

    Figure Lengend Snippet: U1C depletion results in specific alternative splicing alterations in HeLa cells: Specificity and validation. ( A ) U1C knockdown (kd) in HeLa cells. Whole cell lysates were analyzed by SDS-PAGE and Western blot detecting U1C and γ-tubulin. U1 snRNA steady-state levels were analyzed by Northern blotting with probes specific for U1 snRNA and, as a loading control, U3 snoRNA. HeLa cells after U1C knockdown (ΔC) and luciferase-siRNA treated control cells (ctr) were compared. ( B ) Graphical overview of U1C-dependent alternative splicing targets identified by RNA-Seq analysis. ( C ) U1 snRNA blocking in HeLa cells. The efficiency of U1 snRNA blocking was determined by RNase H protection and silver staining. The positions of the full-length U1 snRNA (U1 uncut), the RNase H-cleaved U1 snRNA (U1 cut), and the U2 snRNA (as a control) are marked on the right. ( D ) Alternative splicing patterns of selected U1C target genes (names above the lanes) were analyzed by RT-PCR, using total RNA from HeLa cells after U1C-knockdown (ctr vs. ΔC) or U1 snRNA blocking (ctr vs. U1). Target-specific primers (arrows in the schematics on the right of the panels) were designed to amplify both alternative splicing isoforms. M , DNA size markers (in bp). Upper panel: Top and lower bands represent exon inclusion and skipping products, respectively; an unspecific product for SNHG5 is marked by open circles between the lanes. Lower panel: For MARCH7 top and lower bands reflect usage of the proximal and distal 5′ splice site, respectively. For UFM1 three alternative 5′ splice sites are activated upon U1C knockdown labeled with 1, 2, and 3 on the right.

    Article Snippet: The efficiency of U1 snRNA inhibition was analyzed by an RNase H protection assay: Whole cell extracts were incubated with 5 µM antisense DNA oligonucleotide ( 5′-CAGGTAAGTAT-3′ ) and 1.5 U RNase H (Promega) for 30 min at 37°C.

    Techniques: SDS Page, Western Blot, Northern Blot, Luciferase, RNA Sequencing Assay, Blocking Assay, Silver Staining, Reverse Transcription Polymerase Chain Reaction, Labeling

    Acetylation of GATA-1 causes ubiquitination. ( A ) and β-galactosidase with (+) or without (−) TSA treatment. Lower panel: Western blot showing the levels of ubiquitinated GATA-1 (Ub-GATA-1), acetylated GATA-1 (Ac-GATA-1) and total GATA-1 following immunoprecipitation from transfected Cos 7 cells treated with MG-132 with (+) or without (−) TSA. ( B ) p300 but not p300dHAT decreases GATA-1 levels. NIH3T3 cells were transfected with expression vectors for GATA-1 and p300 or its acetyltransferase mutant, p300dHAT. Whole-cell extracts were used in a Western blot with anti-GATA-1 antibody. β-Galactosidase is the cotransfection control. ( C ). Total GATA-1 levels decrease more in Cos 7 cells (A, upper panel) than BM-SCF cells (C, second panel) upon TSA treatment. GATA-1 regulates its own promoter in haemopoietic cells but is controlled by the EF1α promoter in Cos 7 cells. To prevent TSA increasing GATA-1 levels in BM-SCF cells via GATA-1 autoregulation, we added the transcription inhibitor α-amanitin (α-aman). A more substantial decrease is now seen (fourth panel). An anti-β-tubulin Western confirms that equivalent cell numbers were used (lower panel). ( D ) resulted in a similar increase in GATA-1 stability, suggesting that it is unlikely that loss of ubiquitination sites caused the increased stability. ( E ).

    Journal: The EMBO Journal

    Article Title: Acetylation and MAPK phosphorylation cooperate to regulate the degradation of active GATA-1

    doi: 10.1038/sj.emboj.7601228

    Figure Lengend Snippet: Acetylation of GATA-1 causes ubiquitination. ( A ) and β-galactosidase with (+) or without (−) TSA treatment. Lower panel: Western blot showing the levels of ubiquitinated GATA-1 (Ub-GATA-1), acetylated GATA-1 (Ac-GATA-1) and total GATA-1 following immunoprecipitation from transfected Cos 7 cells treated with MG-132 with (+) or without (−) TSA. ( B ) p300 but not p300dHAT decreases GATA-1 levels. NIH3T3 cells were transfected with expression vectors for GATA-1 and p300 or its acetyltransferase mutant, p300dHAT. Whole-cell extracts were used in a Western blot with anti-GATA-1 antibody. β-Galactosidase is the cotransfection control. ( C ). Total GATA-1 levels decrease more in Cos 7 cells (A, upper panel) than BM-SCF cells (C, second panel) upon TSA treatment. GATA-1 regulates its own promoter in haemopoietic cells but is controlled by the EF1α promoter in Cos 7 cells. To prevent TSA increasing GATA-1 levels in BM-SCF cells via GATA-1 autoregulation, we added the transcription inhibitor α-amanitin (α-aman). A more substantial decrease is now seen (fourth panel). An anti-β-tubulin Western confirms that equivalent cell numbers were used (lower panel). ( D ) resulted in a similar increase in GATA-1 stability, suggesting that it is unlikely that loss of ubiquitination sites caused the increased stability. ( E ).

    Article Snippet: Antibodies were purchased as follows: anti-GATA-1 antibodies N6 (rat monoclonal) and M-20 (goat polyclonal) from Santa Cruz; mouse monoclonal anti-β-galactosidase from Promega; mouse monoclonal anti-ubiquitin (clone FK2) from Affiniti Research; mouse monoclonal anti-tubulin and mouse anti-FLAG (M2) from Sigma.

    Techniques: Western Blot, Immunoprecipitation, Transfection, Expressing, Mutagenesis, Cotransfection

    GATA-1 is ubiquitinated. ( A ) Proteasome inhibitors increase GATA-1 stability. Transfected Cos 7 cells were treated with the proteasome inhibitors shown. Protein levels were analysed by immunoblotting with N6 antibody; β-galactosidase is the cotransfection control. ( B ) Phosphorylation and acetylation affect GATA-1 ubiquitination. Western blot showing the level of ubiquitination of GATA-1 immunoprecipitated from transfected 293T cells. Normalised levels of GATA-1 were loaded; this is verified in the lower panel where the blot is re-probed with a second anti-GATA-1 antibody; densitometry confirmed GATA-1 ratios as wt 1:Ph mut 0.8:Acet mut 0.95. Lanes 5 and 6 are a short exposure to highlight the difference in GATA-1 ubiquitination ±MG-132. Mock-transfected cells confirm that ubiquitinated proteins do not stick nonspecifically during immunoprecipitation.

    Journal: The EMBO Journal

    Article Title: Acetylation and MAPK phosphorylation cooperate to regulate the degradation of active GATA-1

    doi: 10.1038/sj.emboj.7601228

    Figure Lengend Snippet: GATA-1 is ubiquitinated. ( A ) Proteasome inhibitors increase GATA-1 stability. Transfected Cos 7 cells were treated with the proteasome inhibitors shown. Protein levels were analysed by immunoblotting with N6 antibody; β-galactosidase is the cotransfection control. ( B ) Phosphorylation and acetylation affect GATA-1 ubiquitination. Western blot showing the level of ubiquitination of GATA-1 immunoprecipitated from transfected 293T cells. Normalised levels of GATA-1 were loaded; this is verified in the lower panel where the blot is re-probed with a second anti-GATA-1 antibody; densitometry confirmed GATA-1 ratios as wt 1:Ph mut 0.8:Acet mut 0.95. Lanes 5 and 6 are a short exposure to highlight the difference in GATA-1 ubiquitination ±MG-132. Mock-transfected cells confirm that ubiquitinated proteins do not stick nonspecifically during immunoprecipitation.

    Article Snippet: Antibodies were purchased as follows: anti-GATA-1 antibodies N6 (rat monoclonal) and M-20 (goat polyclonal) from Santa Cruz; mouse monoclonal anti-β-galactosidase from Promega; mouse monoclonal anti-ubiquitin (clone FK2) from Affiniti Research; mouse monoclonal anti-tubulin and mouse anti-FLAG (M2) from Sigma.

    Techniques: Transfection, Cotransfection, Western Blot, Immunoprecipitation

    Preferential degradation of transcriptionally active GATA-1. ( A ) Upper: The reporter construct pGL3EpoR. Lower: Inhibition of phosphorylation increases GATA-1-dependent transcription. NIH3T3 cells, transfected with the constructs shown, were treated with or without the MEK inhibitor U0126. The transcription level in the presence of U0126 divided by that in its absence is plotted. The average of four experiments is shown. ( B ) The phosphorylation mutant is more transcriptionally active than wild-type GATA-1. Left panel: S1 mapping of β-globin gene transcription in BM-SCF cells that express FLAG-tagged wild-type (wt) or phosphorylation mutant (ph mut) GATA-1. Quantification of β-globin transcription, normalised to α-actin, is shown beneath the gel. Right panel: Western blot showing the level of the FLAG-tagged wild-type or phosphorylation mutant protein and endogenous GATA-1. For this experiment, cell lines were chosen where mutant and wild-type GATA-1 are expressed at similar levels. ( C ). Control experiments confirmed that transfection efficiency was equivalent. ( D ) The DNA binding mutants are more stable than wild-type GATA-1. Left panel: Western blot of extracts from Cos 7 cells transfected with wild-type GATA-1 and the DNA binding mutants, C261P and RNRK, probed with anti-GATA-1 antibody or anti-acetyl-GATA-1 antibody. β-Galactosidase is the cotransfection control. Right panel: Haemopoietic (BM-SCF) cells, expressing FLAG-tagged RNRK GATA-1, were treated with or without TSA. A Western blot probed with anti-GATA-1 antibody (N-6) is shown.

    Journal: The EMBO Journal

    Article Title: Acetylation and MAPK phosphorylation cooperate to regulate the degradation of active GATA-1

    doi: 10.1038/sj.emboj.7601228

    Figure Lengend Snippet: Preferential degradation of transcriptionally active GATA-1. ( A ) Upper: The reporter construct pGL3EpoR. Lower: Inhibition of phosphorylation increases GATA-1-dependent transcription. NIH3T3 cells, transfected with the constructs shown, were treated with or without the MEK inhibitor U0126. The transcription level in the presence of U0126 divided by that in its absence is plotted. The average of four experiments is shown. ( B ) The phosphorylation mutant is more transcriptionally active than wild-type GATA-1. Left panel: S1 mapping of β-globin gene transcription in BM-SCF cells that express FLAG-tagged wild-type (wt) or phosphorylation mutant (ph mut) GATA-1. Quantification of β-globin transcription, normalised to α-actin, is shown beneath the gel. Right panel: Western blot showing the level of the FLAG-tagged wild-type or phosphorylation mutant protein and endogenous GATA-1. For this experiment, cell lines were chosen where mutant and wild-type GATA-1 are expressed at similar levels. ( C ). Control experiments confirmed that transfection efficiency was equivalent. ( D ) The DNA binding mutants are more stable than wild-type GATA-1. Left panel: Western blot of extracts from Cos 7 cells transfected with wild-type GATA-1 and the DNA binding mutants, C261P and RNRK, probed with anti-GATA-1 antibody or anti-acetyl-GATA-1 antibody. β-Galactosidase is the cotransfection control. Right panel: Haemopoietic (BM-SCF) cells, expressing FLAG-tagged RNRK GATA-1, were treated with or without TSA. A Western blot probed with anti-GATA-1 antibody (N-6) is shown.

    Article Snippet: Antibodies were purchased as follows: anti-GATA-1 antibodies N6 (rat monoclonal) and M-20 (goat polyclonal) from Santa Cruz; mouse monoclonal anti-β-galactosidase from Promega; mouse monoclonal anti-ubiquitin (clone FK2) from Affiniti Research; mouse monoclonal anti-tubulin and mouse anti-FLAG (M2) from Sigma.

    Techniques: Construct, Inhibition, Transfection, Mutagenesis, Western Blot, Binding Assay, Cotransfection, Expressing

    Mutation of phosphorylation and acetylation sites increases GATA-1 stability. ( A ) Upper panels: Western blot showing GATA-1 levels in Cos 7 cells, transfected with constructs to express wild-type GATA-1 (Wt), phosphorylation (Ph) or acetylation (Acet) mutants. β-Galactosidase is the cotransfection control. Lower panels: RNase protection assay of the corresponding GATA-1 mRNAs; the human β-globin gene is the cotransfection control. ( B ) Upper panel: Western blot showing the levels of endogenous and FLAG-tagged GATA-1 protein in BM-SCF cells stably infected with retroviruses to express FLAG-tagged versions of GATA-1. Lower panels: RNase protection assay of the FLAG-tagged and endogenous GATA-1 mRNAs. ( C ) Pulse–chase experiment to determine the half-life of GATA-1. The levels of wild type (⧫), acetylation (Acet ▪) and phosphorylation (Ph ▴) mutants in Cos 7 cells are shown. A value of 100% was given to the amount of protein at time zero; the per cent of protein remaining was calculated relative to this. The plot shows the average of three experiments; error bars show standard error.

    Journal: The EMBO Journal

    Article Title: Acetylation and MAPK phosphorylation cooperate to regulate the degradation of active GATA-1

    doi: 10.1038/sj.emboj.7601228

    Figure Lengend Snippet: Mutation of phosphorylation and acetylation sites increases GATA-1 stability. ( A ) Upper panels: Western blot showing GATA-1 levels in Cos 7 cells, transfected with constructs to express wild-type GATA-1 (Wt), phosphorylation (Ph) or acetylation (Acet) mutants. β-Galactosidase is the cotransfection control. Lower panels: RNase protection assay of the corresponding GATA-1 mRNAs; the human β-globin gene is the cotransfection control. ( B ) Upper panel: Western blot showing the levels of endogenous and FLAG-tagged GATA-1 protein in BM-SCF cells stably infected with retroviruses to express FLAG-tagged versions of GATA-1. Lower panels: RNase protection assay of the FLAG-tagged and endogenous GATA-1 mRNAs. ( C ) Pulse–chase experiment to determine the half-life of GATA-1. The levels of wild type (⧫), acetylation (Acet ▪) and phosphorylation (Ph ▴) mutants in Cos 7 cells are shown. A value of 100% was given to the amount of protein at time zero; the per cent of protein remaining was calculated relative to this. The plot shows the average of three experiments; error bars show standard error.

    Article Snippet: Antibodies were purchased as follows: anti-GATA-1 antibodies N6 (rat monoclonal) and M-20 (goat polyclonal) from Santa Cruz; mouse monoclonal anti-β-galactosidase from Promega; mouse monoclonal anti-ubiquitin (clone FK2) from Affiniti Research; mouse monoclonal anti-tubulin and mouse anti-FLAG (M2) from Sigma.

    Techniques: Mutagenesis, Western Blot, Transfection, Construct, Cotransfection, Rnase Protection Assay, Stable Transfection, Infection, Pulse Chase