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GenScript histone h4
CARM1 represses γ-globin expression. A , Western blot analysis of indicated protein levels in cell lysates from the scramble control (Scr), CARM1-KD1, and CARM1-KD2 Lys-562 cells. GAPDH and <t>histone</t> H4 were used as loading controls. Blots are representative of three independent experiments. B and C , quantitative real-time PCR analysis of CARM1 ( B ) and γ-globin ( C ) mRNA level in Scr, CARM1-KD1, and CARM1-KD2 Lys-562 cells normalized to β-actin mRNA. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. **, p
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1) Product Images from "CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells"

Article Title: CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.004028

CARM1 represses γ-globin expression. A , Western blot analysis of indicated protein levels in cell lysates from the scramble control (Scr), CARM1-KD1, and CARM1-KD2 Lys-562 cells. GAPDH and histone H4 were used as loading controls. Blots are representative of three independent experiments. B and C , quantitative real-time PCR analysis of CARM1 ( B ) and γ-globin ( C ) mRNA level in Scr, CARM1-KD1, and CARM1-KD2 Lys-562 cells normalized to β-actin mRNA. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. **, p
Figure Legend Snippet: CARM1 represses γ-globin expression. A , Western blot analysis of indicated protein levels in cell lysates from the scramble control (Scr), CARM1-KD1, and CARM1-KD2 Lys-562 cells. GAPDH and histone H4 were used as loading controls. Blots are representative of three independent experiments. B and C , quantitative real-time PCR analysis of CARM1 ( B ) and γ-globin ( C ) mRNA level in Scr, CARM1-KD1, and CARM1-KD2 Lys-562 cells normalized to β-actin mRNA. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. **, p

Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

CARM1 methylates PRMT5. A , SDS-PAGE analysis of purified recombinant PRMT5 (rPRMT5) from E. coli as the substrate for following in vitro methylation assays. B , SDS-PAGE analysis of purified recombinant GST-PRMT1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-PRMT1 or GST control ( right ). C , SDS-PAGE analysis of purified recombinant GST-CARM1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H3R17me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-CARM1 or GST control ( right ). A gray asterisk denotes the positive 3 H-labeled PRMT5 band. D , SDS-PAGE analysis of IgG control ( top left ) and of FLAG-PRMT5 immunoprecipitated from Lys-562 cells overexpressing FLAG-tagged PRMT5. SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2s antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with FLAG-PRMT5 or IgG control ( right ). Black asterisks denote the fusion proteins in assays.
Figure Legend Snippet: CARM1 methylates PRMT5. A , SDS-PAGE analysis of purified recombinant PRMT5 (rPRMT5) from E. coli as the substrate for following in vitro methylation assays. B , SDS-PAGE analysis of purified recombinant GST-PRMT1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-PRMT1 or GST control ( right ). C , SDS-PAGE analysis of purified recombinant GST-CARM1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H3R17me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-CARM1 or GST control ( right ). A gray asterisk denotes the positive 3 H-labeled PRMT5 band. D , SDS-PAGE analysis of IgG control ( top left ) and of FLAG-PRMT5 immunoprecipitated from Lys-562 cells overexpressing FLAG-tagged PRMT5. SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2s antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with FLAG-PRMT5 or IgG control ( right ). Black asterisks denote the fusion proteins in assays.

Techniques Used: SDS Page, Purification, Recombinant, In Vitro, Methylation, Staining, Western Blot, Labeling, Immunoprecipitation

Methylation of PRMT5 at Arg-505 is essential for its methyltransferase activity. A , Western blot analysis of extracts from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using FLAG and PRMT5 antibodies. GAPDH was used as a loading control. Blots are representative of three independent experiments. B, quantitative real-time PCR analysis of PRMT5 mRNA normalized to β-actin in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. C , Western blot analysis of extracted histones from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using H4R3me2s antibody. Histone H4 was used as a loading control. Blots are representative of three independent experiments. D , ChIP analysis of H4R3me2s enrichment at the γ-promoter in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. *, p
Figure Legend Snippet: Methylation of PRMT5 at Arg-505 is essential for its methyltransferase activity. A , Western blot analysis of extracts from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using FLAG and PRMT5 antibodies. GAPDH was used as a loading control. Blots are representative of three independent experiments. B, quantitative real-time PCR analysis of PRMT5 mRNA normalized to β-actin in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. C , Western blot analysis of extracted histones from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using H4R3me2s antibody. Histone H4 was used as a loading control. Blots are representative of three independent experiments. D , ChIP analysis of H4R3me2s enrichment at the γ-promoter in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. *, p

Techniques Used: Methylation, Activity Assay, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Two Tailed Test

2) Product Images from "A Conserved Patch near the C Terminus of Histone H4 Is Required for Genome Stability in Budding Yeast ▿"

Article Title: A Conserved Patch near the C Terminus of Histone H4 Is Required for Genome Stability in Budding Yeast ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01432-10

H4 mutants were synthetically sick, with deletion of  MAD2 , a spindle checkpoint gene. Strains YYY67 ( MAD2 ) and YYY85 ( mad2 Δ), carrying a  URA3  plasmid expressing wild-type H4, were transformed with a  TRP1  plasmid expressing wild-type or mutant histone H4, and transformants were selected on SC-Ura-Trp medium. The growth of the transformants was checked after 4 days.
Figure Legend Snippet: H4 mutants were synthetically sick, with deletion of MAD2 , a spindle checkpoint gene. Strains YYY67 ( MAD2 ) and YYY85 ( mad2 Δ), carrying a URA3 plasmid expressing wild-type H4, were transformed with a TRP1 plasmid expressing wild-type or mutant histone H4, and transformants were selected on SC-Ura-Trp medium. The growth of the transformants was checked after 4 days.

Techniques Used: Plasmid Preparation, Expressing, Transformation Assay, Mutagenesis

Model for the deposition defect caused by the histone H4 mutants. (A) Rtt106 or CAF-I deposits wild-type H4/H3 dimers (or possibly tetramers) onto DNA. (B) Mutant (MT) H4/H3 dimers accumulate on Rtt106 or CAF-I and cannot be transferred to the next step. Poor histone deposition causes a lower nucleosome density on the chromatin. (C) In the cells expressing both wild-type and mutant histone H4, the deposition of wild-type histone H4 is also affected because a certain fraction of the chaperone becomes inactive by nonproductive binding of mutant H4. (D) In cells with RTT106 , CAC1 , or CAC2 deletions, mutant H4/H3 cannot accumulate on either of the chaperones and thus can be transferred onto DNA more efficiently by another chaperone (colored purple).
Figure Legend Snippet: Model for the deposition defect caused by the histone H4 mutants. (A) Rtt106 or CAF-I deposits wild-type H4/H3 dimers (or possibly tetramers) onto DNA. (B) Mutant (MT) H4/H3 dimers accumulate on Rtt106 or CAF-I and cannot be transferred to the next step. Poor histone deposition causes a lower nucleosome density on the chromatin. (C) In the cells expressing both wild-type and mutant histone H4, the deposition of wild-type histone H4 is also affected because a certain fraction of the chaperone becomes inactive by nonproductive binding of mutant H4. (D) In cells with RTT106 , CAC1 , or CAC2 deletions, mutant H4/H3 cannot accumulate on either of the chaperones and thus can be transferred onto DNA more efficiently by another chaperone (colored purple).

Techniques Used: Mutagenesis, Expressing, Binding Assay

Genome instability of the H4 mutants. (A) Chromosome III loss frequency of the H4 mutants. MAT α cells expressing wild-type or mutant histone H4 were tested for the capacity to mate with a MAT α test strain. The loss frequency is the number of cells that were able to mate divided by the total number of cells. The average of 3 independent experiments is shown. The error bars indicate standard deviations. (B) CEN plasmid loss frequency of the H4 mutants. Strains containing a CEN LEU2 plasmid (pRS315) and expressing wild-type or mutant H4 were used to measure the plasmid loss frequency as described in Materials and Methods. The average of 4 experiments is shown.
Figure Legend Snippet: Genome instability of the H4 mutants. (A) Chromosome III loss frequency of the H4 mutants. MAT α cells expressing wild-type or mutant histone H4 were tested for the capacity to mate with a MAT α test strain. The loss frequency is the number of cells that were able to mate divided by the total number of cells. The average of 3 independent experiments is shown. The error bars indicate standard deviations. (B) CEN plasmid loss frequency of the H4 mutants. Strains containing a CEN LEU2 plasmid (pRS315) and expressing wild-type or mutant H4 were used to measure the plasmid loss frequency as described in Materials and Methods. The average of 4 experiments is shown.

Techniques Used: Expressing, Mutagenesis, Plasmid Preparation

H4 mutants displayed altered chromatin structure. (A) The sensitivities of nuclei to digestion by the restriction enzyme DraI at the CEN3 locus were compared for the wild type and the H4 mutants. A schematic representation of an EcoRI fragment containing CEN3 and its CDE II element containing three DraI sites is depicted. The sensitivity of these sites to DraI digestion of nuclei was measured by Southern blotting using a 0.9-kb probe located downstream of CEN3 . DNA fragments generated by DraI digestion from these closely spaced sites were not separated from each other on the agarose gel used. The percentage of the CEN3 EcoRI fragment cut by DraI was calculated from the intensity of the 2.9-kb fragment compared to the intensity of the intact 5.1-kb fragment plus the 2.9-kb fragment and is shown on the right for the wild type and the H4 mutants. (B) A measurement of DraI sensitivity similar to that in panel A was performed for the GAL10 locus. In this case, an AvaI-NdeI fragment containing a single DraI site was used. The GAL10 locus is depicted on the left, and the percent DraI digestion is shown on the right. (C) Chromatin in H4 mutants was more accessible for micrococcal nuclease (MNase) digestion. Nuclei were purified from the cells expressing wild-type or mutant histone H4 and treated with MNase for 10, 20, 30, or 40 min. DNA was purified, resolved by agarose electrophoresis, and visualized by ethidium bromide staining (left). The intensities of the bands of mono-, di-, or trinucleosomes for the 30-min samples were quantified. The amount of trinucleosomes was designated arbitrary unit 1. The relative amounts of mononucleosomes and di-nucleosomes are shown in the graph on the right. Experiments were repeated in triplicate. (D) Chromatin immunoprecipitation of H4 (WT and Y98A) at three different loci. The percentage is the signal relative to the input and is an average of three independent experiments. The error bars indicate standard deviations.
Figure Legend Snippet: H4 mutants displayed altered chromatin structure. (A) The sensitivities of nuclei to digestion by the restriction enzyme DraI at the CEN3 locus were compared for the wild type and the H4 mutants. A schematic representation of an EcoRI fragment containing CEN3 and its CDE II element containing three DraI sites is depicted. The sensitivity of these sites to DraI digestion of nuclei was measured by Southern blotting using a 0.9-kb probe located downstream of CEN3 . DNA fragments generated by DraI digestion from these closely spaced sites were not separated from each other on the agarose gel used. The percentage of the CEN3 EcoRI fragment cut by DraI was calculated from the intensity of the 2.9-kb fragment compared to the intensity of the intact 5.1-kb fragment plus the 2.9-kb fragment and is shown on the right for the wild type and the H4 mutants. (B) A measurement of DraI sensitivity similar to that in panel A was performed for the GAL10 locus. In this case, an AvaI-NdeI fragment containing a single DraI site was used. The GAL10 locus is depicted on the left, and the percent DraI digestion is shown on the right. (C) Chromatin in H4 mutants was more accessible for micrococcal nuclease (MNase) digestion. Nuclei were purified from the cells expressing wild-type or mutant histone H4 and treated with MNase for 10, 20, 30, or 40 min. DNA was purified, resolved by agarose electrophoresis, and visualized by ethidium bromide staining (left). The intensities of the bands of mono-, di-, or trinucleosomes for the 30-min samples were quantified. The amount of trinucleosomes was designated arbitrary unit 1. The relative amounts of mononucleosomes and di-nucleosomes are shown in the graph on the right. Experiments were repeated in triplicate. (D) Chromatin immunoprecipitation of H4 (WT and Y98A) at three different loci. The percentage is the signal relative to the input and is an average of three independent experiments. The error bars indicate standard deviations.

Techniques Used: Southern Blot, Generated, Agarose Gel Electrophoresis, Purification, Expressing, Mutagenesis, Electrophoresis, Staining, Chromatin Immunoprecipitation

Interaction of histone chaperones Rtt106 and CAF-I with H4. (A) Rtt106 purification. Rtt106-TAP was purified from the cells expressing wild-type or mutant H4. Copurified proteins were detected with Western blots using antibodies against H4, H3, H3K56Ac, Cac2, and Rtt106 by anti-CBP antibody. The antibodies were also used to detect protein levels in the whole-cell extract (WCE). (B) CAF-I purification using TAP-tagged Cac2 from cells expressing wild-type or mutant H4. Copurified proteins were detected by Western blots using antibodies to H4 and H3. Cac2 was detected with an anti-CBP antibody. (C) In vitro binding of histones purified from yeast to Rtt106. Wild-type or H4 mutant histones were purified from yeast, resolved by SDS-PAGE, and visualized by Coomassie blue (CB) staining (top) or by Western blotting (bottom). H3* is an N-terminally truncated form of H3 commonly found in yeast extracts. The histones were used in a pulldown experiment with GST-Rtt106 as described in Materials and Methods. Two concentrations of histones were mixed with 36 pmol of GST-Rtt106. (D) In vitro binding of recombinant H4 and H3 to Rtt106. Wild-type or Y98 mutant H4, along with H3, was coexpressed and purified from E. coli . H4 and H3 were used in a pulldown experiment with GST-Rtt106. Three concentrations of histones were mixed with 30 pmol of Rtt106.
Figure Legend Snippet: Interaction of histone chaperones Rtt106 and CAF-I with H4. (A) Rtt106 purification. Rtt106-TAP was purified from the cells expressing wild-type or mutant H4. Copurified proteins were detected with Western blots using antibodies against H4, H3, H3K56Ac, Cac2, and Rtt106 by anti-CBP antibody. The antibodies were also used to detect protein levels in the whole-cell extract (WCE). (B) CAF-I purification using TAP-tagged Cac2 from cells expressing wild-type or mutant H4. Copurified proteins were detected by Western blots using antibodies to H4 and H3. Cac2 was detected with an anti-CBP antibody. (C) In vitro binding of histones purified from yeast to Rtt106. Wild-type or H4 mutant histones were purified from yeast, resolved by SDS-PAGE, and visualized by Coomassie blue (CB) staining (top) or by Western blotting (bottom). H3* is an N-terminally truncated form of H3 commonly found in yeast extracts. The histones were used in a pulldown experiment with GST-Rtt106 as described in Materials and Methods. Two concentrations of histones were mixed with 36 pmol of GST-Rtt106. (D) In vitro binding of recombinant H4 and H3 to Rtt106. Wild-type or Y98 mutant H4, along with H3, was coexpressed and purified from E. coli . H4 and H3 were used in a pulldown experiment with GST-Rtt106. Three concentrations of histones were mixed with 30 pmol of Rtt106.

Techniques Used: Purification, Expressing, Mutagenesis, Western Blot, In Vitro, Binding Assay, SDS Page, Staining, Recombinant

3) Product Images from "CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells"

Article Title: CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.004028

CARM1 represses γ-globin expression. A , Western blot analysis of indicated protein levels in cell lysates from the scramble control (Scr), CARM1-KD1, and CARM1-KD2 Lys-562 cells. GAPDH and histone H4 were used as loading controls. Blots are representative of three independent experiments. B and C , quantitative real-time PCR analysis of CARM1 ( B ) and γ-globin ( C ) mRNA level in Scr, CARM1-KD1, and CARM1-KD2 Lys-562 cells normalized to β-actin mRNA. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. **, p
Figure Legend Snippet: CARM1 represses γ-globin expression. A , Western blot analysis of indicated protein levels in cell lysates from the scramble control (Scr), CARM1-KD1, and CARM1-KD2 Lys-562 cells. GAPDH and histone H4 were used as loading controls. Blots are representative of three independent experiments. B and C , quantitative real-time PCR analysis of CARM1 ( B ) and γ-globin ( C ) mRNA level in Scr, CARM1-KD1, and CARM1-KD2 Lys-562 cells normalized to β-actin mRNA. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. **, p

Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

CARM1 methylates PRMT5. A , SDS-PAGE analysis of purified recombinant PRMT5 (rPRMT5) from E. coli as the substrate for following in vitro methylation assays. B , SDS-PAGE analysis of purified recombinant GST-PRMT1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-PRMT1 or GST control ( right ). C , SDS-PAGE analysis of purified recombinant GST-CARM1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H3R17me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-CARM1 or GST control ( right ). A gray asterisk denotes the positive 3 H-labeled PRMT5 band. D , SDS-PAGE analysis of IgG control ( top left ) and of FLAG-PRMT5 immunoprecipitated from Lys-562 cells overexpressing FLAG-tagged PRMT5. SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2s antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with FLAG-PRMT5 or IgG control ( right ). Black asterisks denote the fusion proteins in assays.
Figure Legend Snippet: CARM1 methylates PRMT5. A , SDS-PAGE analysis of purified recombinant PRMT5 (rPRMT5) from E. coli as the substrate for following in vitro methylation assays. B , SDS-PAGE analysis of purified recombinant GST-PRMT1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-PRMT1 or GST control ( right ). C , SDS-PAGE analysis of purified recombinant GST-CARM1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H3R17me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-CARM1 or GST control ( right ). A gray asterisk denotes the positive 3 H-labeled PRMT5 band. D , SDS-PAGE analysis of IgG control ( top left ) and of FLAG-PRMT5 immunoprecipitated from Lys-562 cells overexpressing FLAG-tagged PRMT5. SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2s antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with FLAG-PRMT5 or IgG control ( right ). Black asterisks denote the fusion proteins in assays.

Techniques Used: SDS Page, Purification, Recombinant, In Vitro, Methylation, Staining, Western Blot, Labeling, Immunoprecipitation

Methylation of PRMT5 at Arg-505 is essential for its methyltransferase activity. A , Western blot analysis of extracts from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using FLAG and PRMT5 antibodies. GAPDH was used as a loading control. Blots are representative of three independent experiments. B, quantitative real-time PCR analysis of PRMT5 mRNA normalized to β-actin in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. C , Western blot analysis of extracted histones from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using H4R3me2s antibody. Histone H4 was used as a loading control. Blots are representative of three independent experiments. D , ChIP analysis of H4R3me2s enrichment at the γ-promoter in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. *, p
Figure Legend Snippet: Methylation of PRMT5 at Arg-505 is essential for its methyltransferase activity. A , Western blot analysis of extracts from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using FLAG and PRMT5 antibodies. GAPDH was used as a loading control. Blots are representative of three independent experiments. B, quantitative real-time PCR analysis of PRMT5 mRNA normalized to β-actin in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. C , Western blot analysis of extracted histones from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using H4R3me2s antibody. Histone H4 was used as a loading control. Blots are representative of three independent experiments. D , ChIP analysis of H4R3me2s enrichment at the γ-promoter in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. *, p

Techniques Used: Methylation, Activity Assay, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Two Tailed Test

Related Articles

Western Blot:

Article Title: A Conserved Patch near the C Terminus of Histone H4 Is Required for Genome Stability in Budding Yeast ▿
Article Snippet: .. The bound proteins were eluted with Laemmli buffer, resolved by SDS-PAGE, and detected by Western blotting using antibodies against histone H3 (Abcam; ab1791), histone H4 (from the laboratory of A. Shilatifard), H3 K56Ac (from Zhiguo Zhang), or CBP tag (GenScript; ). .. To purify histones from yeast, strain YYY67 was transformed with plasmids expressing WT H4 or each of the four mutants, and transformants were selected on SC-Trp medium and counterselected on SC-Trp plus 5-FOA.

Article Title: PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks
Article Snippet: .. Antibodies and western blot Antibodies obtained from commercial sources were as following: β-actin and Histone H3 from Genscript, Ku86 (C-20) and p53 (DO-1) from Santa Cruz Biotechnology, 53BP1 (Ab172580) and NBS1 (Ab175800) from Abcam, pSer139-H2AX (clone: JWB301), BRCA1 (clone MS110) from Merck-Millipore, pSer345-CHK1 and PHF2 from Cell Signalling, PHF2 and pSer4/8-RPA2 from Bethyl, RPA2 from Novus Biologicals, Rad51 by Invitrogen and CtIP from Active Motif. .. Also, antibodies against CtIP and Mre11 were generated by injecting rabbits with a His-tagged antigen (amino acids 150–500 and 182–480, respectively) that were obtained by expression in bacteria and purification with a Ni-NTA resin (Qiagen) following manufacturers recommendations.

Article Title: CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells
Article Snippet: .. The following antibodies were used for Western blotting: FLAG (Sigma-Aldrich), PRMT5 (Sigma-Aldrich), CARM1 (Cell Signaling Technology), GAPDH (MBL International), H4R3me2s (Abcam), H4R3me2a (Active Motif), H3R17me2a (Abcam), histone H4 (GenScript), and histone H3 (GenScript). .. Human PRMT1, CARM1, and full-length or truncated PRMT5 cDNA sequences were cloned into a pGEX6p-1 vector.

SDS Page:

Article Title: A Conserved Patch near the C Terminus of Histone H4 Is Required for Genome Stability in Budding Yeast ▿
Article Snippet: .. The bound proteins were eluted with Laemmli buffer, resolved by SDS-PAGE, and detected by Western blotting using antibodies against histone H3 (Abcam; ab1791), histone H4 (from the laboratory of A. Shilatifard), H3 K56Ac (from Zhiguo Zhang), or CBP tag (GenScript; ). .. To purify histones from yeast, strain YYY67 was transformed with plasmids expressing WT H4 or each of the four mutants, and transformants were selected on SC-Trp medium and counterselected on SC-Trp plus 5-FOA.

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    GenScript rare luc dna
    The role of E 3 ubiquitin ligase activity (Panels A and B) and histone deacetylase activity (Panel C) in the repression of RARβ 3 transcriptional activity by HACE1 A. GST-pulldown assays were performed with wild type GST-SF HACE 1 or GST-SF HACE1 C529A and in vitro transcribed and translated [ 35 S]-methionine labeled RARβ 3 . B. Cos1 cells were co-transfected with <t>DNA</t> constructs including HACE1 expression plasmid (pCMVXL4-LF HACE1), HACE1 CA mutant expression plasmid (pCMVXL4-LF HACE1 C876A) or empty plasmid along with <t>pTL-RARE-luc</t> reporter plasmid, pRL reporter plasmid, and RARβ 3 expression plasmid (pOPRSVICAT- RARβ 3 ) or empty expression plasmid. C. Cos1 cells were cotransfected with expression plasmid of LF HACE1 (pCMVXL4-LF HACE1) or empty expression plasmid, pTL-RARE-luc reporter plasmid, pRL reporter plasmid and expression plasmid of RARβ 3 . Twenty-four hr after transfection, cells were treated with combinations of ethanol/10 -6 M RA and 100ng/ml TSA for an additional 24 hr. For panel B, the fold changes were calculated relative to cells that were transfected with empty expression plasmid DNA and treated with ethanol arbitrarily set to 1. For panel C, the fold changes were calculated relative to cells that were transfected with empty expression plasmid DNA, treated with ethanol and no TSA arbitrarily set to 1. For panels B and C, values are the Mean + SD of 3 independent experiments performed in triplicate.
    Rare Luc Dna, supplied by GenScript, 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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    GenScript anti histone h3
    Identification of YAP/TEAD as previously unknown ERα-interacting cofactors by proximity BioID proteomics (A) Schematic diagram demonstrating BioID (proximity-dependent biotin identification) approach for identification of ERα-interacting nuclear proteins. (B) Western blots confirming the inducible expression and in vivo biotinylation in the established ERα-BioID tet-on stable cell line. The fractionation of cytoplasmic (Cyto) and nuclear (Nuc) fractions of MCF7 cells was confirmed with Western blots for GAPDH (cytoplasm-specific marker) and <t>Histone</t> H3 (nucleus-specific marker). The doxycycline-inducible ERα-BirA*-HA fusion protein expression was detected by antibodies recognizing HA and ERα (the endogenous ERα was labeled with * and the tagged exogenous ERα was labeled with #). Biotinylated proteins by ERα-BirA* were detected by streptavidin-HRP blot. (C) Identification of ERα-interacting cofactors by mass spectrometry analyses on the protein complex pulled down from the nuclear fractions of ERα-BioID tet-on stable cell line under the indicated treatments. Biotinylated proteins in nuclear fraction were enriched and purified using streptavidin beads before subjected to mass spectrometry. Besides many listed known cofactors, YAP1 and TEAD4 are two previously unknown ERα-interacting cofactors identified from our studies. Peptide numbers detected from mass spectrometry analyses are listed in the table for each protein. (D) Co-IP assays in MCF7 cells with the indicated treatments confirming protein-protein interactions of endogenous ERα, YAP1 and TEAD4. Nuclear fractions treated with vehicle or E 2 treatment were used for immunoprecipitation with antibodies against ERα, TEAD4 and YAP1 respectively. (E) Western blots confirming the inducible expression and in vivo biotinylation in the established FOXA1-BioID tet-on stable cell line. The fractionation of cytoplasmic (Cyto) and nuclear (Nuc) fractions of MCF7 cells was confirmed with Western blots for GAPDH and Histone H3 respectively. The doxycycline-inducible Myc-BirA*-FOXA1 fusion protein expression was detected by antibodies recognizing Myc and FOXA1 (the endogenous FOXA1 was labeled with * and the tagged exogenous FOXA1 was labeled with #). Biotinylated proteins by BirA*-FOXA1 were detected by streptavidin-HRP blot. (F) Identification of FOXA1-interacting factors by mass spectrometry analyses on streptavidin bead pulldowns from the nuclear fractions of FOXA1-BioID tet-on stable cell line with the indicated treatments. Peptide numbers detected from mass spectrometry analyses are listed in the table. .
    Anti Histone H3, supplied by GenScript, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenScript full length gallus gallus parp3
    Mechanism of DNA break binding by the cPARP3 WGR domain. ( a ) Schematic representation of <t>PARP3</t> domains with the human (top) and chicken (bottom) amino acid positions indicated. The WGR domain is shown in expanded format below showing the HSQC perturbed residues of chicken PARP3 (bottom) and the equivalent residues in human PARP3 (top). ( b ) Overlay of 1 H– 15 N HSQC NMR full spectra for cPARP3 1–169 in the absence (blue) or presence (red) of oligodeoxyribonucleotide duplex harbouring a 5′-phosphorylated nick (protein:DNA ratios of 1:0 and 1:2, respectively). ( c ) Expanded view of the small boxed region shown in b demonstrating the chemical shifts induced in cPARP3 1–169 by different concentrations of nicked DNA. Protein:DNA ratios were 1:0 (that is, no DNA; blue), 5:1 (magenta), 1:1 (green) and 1:2 (red). ( d ) Map of significant chemical shifts induced in cPARP3 1–169 by DNA duplex harbouring a 5′-phosphorylated nick ( > 0.1 p.p.m) or 10-bp 3′-overhang with a recessed 5′-phosphorylated terminus ( > 0.04), surface modelled using CS-Rosetta 41 . Residues with a significant chemical shift in the presence of either a nick (blue) or 3′-overhang (green) or both (red) are indicated. ( e ) Electrostatic surface of modelled cPARP31–169 with nicked DNA. ( f ) Model of cPARP3 1–169 with nicked DNA, depicting residues with significant chemical shifts as above. ( g ) Model of cPARP3 1–169 with nicked DNA lacking the strand located upstream (5′) of the nick (that is, harbouring a DSB with 10-bp 3′-overhang). Residues exhibiting a significant chemical shift are indicated as above.
    Full Length Gallus Gallus Parp3, supplied by GenScript, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenScript histone h3
    CARM1 represses γ-globin expression. A , Western blot analysis of indicated protein levels in cell lysates from the scramble control (Scr), CARM1-KD1, and CARM1-KD2 Lys-562 cells. GAPDH and <t>histone</t> H4 were used as loading controls. Blots are representative of three independent experiments. B and C , quantitative real-time PCR analysis of CARM1 ( B ) and γ-globin ( C ) mRNA level in Scr, CARM1-KD1, and CARM1-KD2 Lys-562 cells normalized to β-actin mRNA. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. **, p
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    Image Search Results


    The role of E 3 ubiquitin ligase activity (Panels A and B) and histone deacetylase activity (Panel C) in the repression of RARβ 3 transcriptional activity by HACE1 A. GST-pulldown assays were performed with wild type GST-SF HACE 1 or GST-SF HACE1 C529A and in vitro transcribed and translated [ 35 S]-methionine labeled RARβ 3 . B. Cos1 cells were co-transfected with DNA constructs including HACE1 expression plasmid (pCMVXL4-LF HACE1), HACE1 CA mutant expression plasmid (pCMVXL4-LF HACE1 C876A) or empty plasmid along with pTL-RARE-luc reporter plasmid, pRL reporter plasmid, and RARβ 3 expression plasmid (pOPRSVICAT- RARβ 3 ) or empty expression plasmid. C. Cos1 cells were cotransfected with expression plasmid of LF HACE1 (pCMVXL4-LF HACE1) or empty expression plasmid, pTL-RARE-luc reporter plasmid, pRL reporter plasmid and expression plasmid of RARβ 3 . Twenty-four hr after transfection, cells were treated with combinations of ethanol/10 -6 M RA and 100ng/ml TSA for an additional 24 hr. For panel B, the fold changes were calculated relative to cells that were transfected with empty expression plasmid DNA and treated with ethanol arbitrarily set to 1. For panel C, the fold changes were calculated relative to cells that were transfected with empty expression plasmid DNA, treated with ethanol and no TSA arbitrarily set to 1. For panels B and C, values are the Mean + SD of 3 independent experiments performed in triplicate.

    Journal: Journal of cellular biochemistry

    Article Title: HACE1: A Novel Repressor of RAR Transcriptional Activity

    doi: 10.1002/jcb.22146

    Figure Lengend Snippet: The role of E 3 ubiquitin ligase activity (Panels A and B) and histone deacetylase activity (Panel C) in the repression of RARβ 3 transcriptional activity by HACE1 A. GST-pulldown assays were performed with wild type GST-SF HACE 1 or GST-SF HACE1 C529A and in vitro transcribed and translated [ 35 S]-methionine labeled RARβ 3 . B. Cos1 cells were co-transfected with DNA constructs including HACE1 expression plasmid (pCMVXL4-LF HACE1), HACE1 CA mutant expression plasmid (pCMVXL4-LF HACE1 C876A) or empty plasmid along with pTL-RARE-luc reporter plasmid, pRL reporter plasmid, and RARβ 3 expression plasmid (pOPRSVICAT- RARβ 3 ) or empty expression plasmid. C. Cos1 cells were cotransfected with expression plasmid of LF HACE1 (pCMVXL4-LF HACE1) or empty expression plasmid, pTL-RARE-luc reporter plasmid, pRL reporter plasmid and expression plasmid of RARβ 3 . Twenty-four hr after transfection, cells were treated with combinations of ethanol/10 -6 M RA and 100ng/ml TSA for an additional 24 hr. For panel B, the fold changes were calculated relative to cells that were transfected with empty expression plasmid DNA and treated with ethanol arbitrarily set to 1. For panel C, the fold changes were calculated relative to cells that were transfected with empty expression plasmid DNA, treated with ethanol and no TSA arbitrarily set to 1. For panels B and C, values are the Mean + SD of 3 independent experiments performed in triplicate.

    Article Snippet: Cos1 cells were cotransfected with V5-HACE1 or GST-HACE1 expression plasmid DNA, GST-RARβ3 or V5-RARβ3 plasmid DNA, RXRα-pSG5 DNA and RARE-luc DNA by GenJet transfection method (Genscript).

    Techniques: Activity Assay, Histone Deacetylase Assay, In Vitro, Labeling, Transfection, Construct, Expressing, Plasmid Preparation, Mutagenesis

    Analysis of RARβ 3 half life in the absence and presence of HACE1 Cos1 cells were transfected with V5-LF HACE1 DNA or empty vector DNA, V5-RARβ 3 DNA, RXRα DNA and RARE-luc DNA. Cells were treated with 10 μg/ml cycloheximide, and 10 -6 M RA or ethanol for the indicated times. (A). Whole cell extracts were prepared from cells with indicated times of treatment and western blots were performed using V5 or GAPDH primary antibodies, donkey anti-mouse IRdye 800CW or donkey anti-rabbit IRDye 680CW secondary antibodies, and detected using a LI-COR Odyssey instrument. GAPDH was used as a loading control. A representative figure from one of three independent experiments is shown. (B). Densitometric values of each band from western blots were quantitated using LI-COR software. V5-RARβ 3 and V5-HACE1 values were normalized with corresponding GAPDH values. The normalized values of V5-RARβ 3 and V5-HACE1 were plotted. A representative plot from one of three independent experiments is shown, time 0 was set to 1 arbitrarily. (C). Half life (protein level = 50% of time 0) of RARβ 3 was calculated based on the linear equations generated from quantitated protein density over time from three independent experiments. Values are the Mean ± SD of 3 independent experiments. P value was generated by pairwise student T test, *P

    Journal: Journal of cellular biochemistry

    Article Title: HACE1: A Novel Repressor of RAR Transcriptional Activity

    doi: 10.1002/jcb.22146

    Figure Lengend Snippet: Analysis of RARβ 3 half life in the absence and presence of HACE1 Cos1 cells were transfected with V5-LF HACE1 DNA or empty vector DNA, V5-RARβ 3 DNA, RXRα DNA and RARE-luc DNA. Cells were treated with 10 μg/ml cycloheximide, and 10 -6 M RA or ethanol for the indicated times. (A). Whole cell extracts were prepared from cells with indicated times of treatment and western blots were performed using V5 or GAPDH primary antibodies, donkey anti-mouse IRdye 800CW or donkey anti-rabbit IRDye 680CW secondary antibodies, and detected using a LI-COR Odyssey instrument. GAPDH was used as a loading control. A representative figure from one of three independent experiments is shown. (B). Densitometric values of each band from western blots were quantitated using LI-COR software. V5-RARβ 3 and V5-HACE1 values were normalized with corresponding GAPDH values. The normalized values of V5-RARβ 3 and V5-HACE1 were plotted. A representative plot from one of three independent experiments is shown, time 0 was set to 1 arbitrarily. (C). Half life (protein level = 50% of time 0) of RARβ 3 was calculated based on the linear equations generated from quantitated protein density over time from three independent experiments. Values are the Mean ± SD of 3 independent experiments. P value was generated by pairwise student T test, *P

    Article Snippet: Cos1 cells were cotransfected with V5-HACE1 or GST-HACE1 expression plasmid DNA, GST-RARβ3 or V5-RARβ3 plasmid DNA, RXRα-pSG5 DNA and RARE-luc DNA by GenJet transfection method (Genscript).

    Techniques: Transfection, Plasmid Preparation, Western Blot, Software, Generated

    Identification of YAP/TEAD as previously unknown ERα-interacting cofactors by proximity BioID proteomics (A) Schematic diagram demonstrating BioID (proximity-dependent biotin identification) approach for identification of ERα-interacting nuclear proteins. (B) Western blots confirming the inducible expression and in vivo biotinylation in the established ERα-BioID tet-on stable cell line. The fractionation of cytoplasmic (Cyto) and nuclear (Nuc) fractions of MCF7 cells was confirmed with Western blots for GAPDH (cytoplasm-specific marker) and Histone H3 (nucleus-specific marker). The doxycycline-inducible ERα-BirA*-HA fusion protein expression was detected by antibodies recognizing HA and ERα (the endogenous ERα was labeled with * and the tagged exogenous ERα was labeled with #). Biotinylated proteins by ERα-BirA* were detected by streptavidin-HRP blot. (C) Identification of ERα-interacting cofactors by mass spectrometry analyses on the protein complex pulled down from the nuclear fractions of ERα-BioID tet-on stable cell line under the indicated treatments. Biotinylated proteins in nuclear fraction were enriched and purified using streptavidin beads before subjected to mass spectrometry. Besides many listed known cofactors, YAP1 and TEAD4 are two previously unknown ERα-interacting cofactors identified from our studies. Peptide numbers detected from mass spectrometry analyses are listed in the table for each protein. (D) Co-IP assays in MCF7 cells with the indicated treatments confirming protein-protein interactions of endogenous ERα, YAP1 and TEAD4. Nuclear fractions treated with vehicle or E 2 treatment were used for immunoprecipitation with antibodies against ERα, TEAD4 and YAP1 respectively. (E) Western blots confirming the inducible expression and in vivo biotinylation in the established FOXA1-BioID tet-on stable cell line. The fractionation of cytoplasmic (Cyto) and nuclear (Nuc) fractions of MCF7 cells was confirmed with Western blots for GAPDH and Histone H3 respectively. The doxycycline-inducible Myc-BirA*-FOXA1 fusion protein expression was detected by antibodies recognizing Myc and FOXA1 (the endogenous FOXA1 was labeled with * and the tagged exogenous FOXA1 was labeled with #). Biotinylated proteins by BirA*-FOXA1 were detected by streptavidin-HRP blot. (F) Identification of FOXA1-interacting factors by mass spectrometry analyses on streptavidin bead pulldowns from the nuclear fractions of FOXA1-BioID tet-on stable cell line with the indicated treatments. Peptide numbers detected from mass spectrometry analyses are listed in the table. .

    Journal: Molecular cell

    Article Title: A non-canonical role of YAP/TEAD is required for activation of estrogen-regulated enhancers in breast cancer

    doi: 10.1016/j.molcel.2019.06.010

    Figure Lengend Snippet: Identification of YAP/TEAD as previously unknown ERα-interacting cofactors by proximity BioID proteomics (A) Schematic diagram demonstrating BioID (proximity-dependent biotin identification) approach for identification of ERα-interacting nuclear proteins. (B) Western blots confirming the inducible expression and in vivo biotinylation in the established ERα-BioID tet-on stable cell line. The fractionation of cytoplasmic (Cyto) and nuclear (Nuc) fractions of MCF7 cells was confirmed with Western blots for GAPDH (cytoplasm-specific marker) and Histone H3 (nucleus-specific marker). The doxycycline-inducible ERα-BirA*-HA fusion protein expression was detected by antibodies recognizing HA and ERα (the endogenous ERα was labeled with * and the tagged exogenous ERα was labeled with #). Biotinylated proteins by ERα-BirA* were detected by streptavidin-HRP blot. (C) Identification of ERα-interacting cofactors by mass spectrometry analyses on the protein complex pulled down from the nuclear fractions of ERα-BioID tet-on stable cell line under the indicated treatments. Biotinylated proteins in nuclear fraction were enriched and purified using streptavidin beads before subjected to mass spectrometry. Besides many listed known cofactors, YAP1 and TEAD4 are two previously unknown ERα-interacting cofactors identified from our studies. Peptide numbers detected from mass spectrometry analyses are listed in the table for each protein. (D) Co-IP assays in MCF7 cells with the indicated treatments confirming protein-protein interactions of endogenous ERα, YAP1 and TEAD4. Nuclear fractions treated with vehicle or E 2 treatment were used for immunoprecipitation with antibodies against ERα, TEAD4 and YAP1 respectively. (E) Western blots confirming the inducible expression and in vivo biotinylation in the established FOXA1-BioID tet-on stable cell line. The fractionation of cytoplasmic (Cyto) and nuclear (Nuc) fractions of MCF7 cells was confirmed with Western blots for GAPDH and Histone H3 respectively. The doxycycline-inducible Myc-BirA*-FOXA1 fusion protein expression was detected by antibodies recognizing Myc and FOXA1 (the endogenous FOXA1 was labeled with * and the tagged exogenous FOXA1 was labeled with #). Biotinylated proteins by BirA*-FOXA1 were detected by streptavidin-HRP blot. (F) Identification of FOXA1-interacting factors by mass spectrometry analyses on streptavidin bead pulldowns from the nuclear fractions of FOXA1-BioID tet-on stable cell line with the indicated treatments. Peptide numbers detected from mass spectrometry analyses are listed in the table. .

    Article Snippet: The antibodies used in this assay were: anti-ERα (sc-543, Santa Cruz), anti-YAP1 (14074S, Cell Signaling Technology), anti-TEAD4 (sc-101184, Santa Cruz), anti-Histone H3 (A01502, GenScript), antiHA (ab9110, Abcam), anti-Myc (2276S, Cell Signaling Technology), anti-FOXA1 (ab5089, Abcam), anti-TAZ (sc-293183, Santa Cruz), anti-Flag (F1804, Sigma) and anti-GAPDH (sc-25778, Santa Cruz).

    Techniques: Western Blot, Expressing, In Vivo, Stable Transfection, Fractionation, Marker, Labeling, Mass Spectrometry, Purification, Co-Immunoprecipitation Assay, Immunoprecipitation

    Mechanism of DNA break binding by the cPARP3 WGR domain. ( a ) Schematic representation of PARP3 domains with the human (top) and chicken (bottom) amino acid positions indicated. The WGR domain is shown in expanded format below showing the HSQC perturbed residues of chicken PARP3 (bottom) and the equivalent residues in human PARP3 (top). ( b ) Overlay of 1 H– 15 N HSQC NMR full spectra for cPARP3 1–169 in the absence (blue) or presence (red) of oligodeoxyribonucleotide duplex harbouring a 5′-phosphorylated nick (protein:DNA ratios of 1:0 and 1:2, respectively). ( c ) Expanded view of the small boxed region shown in b demonstrating the chemical shifts induced in cPARP3 1–169 by different concentrations of nicked DNA. Protein:DNA ratios were 1:0 (that is, no DNA; blue), 5:1 (magenta), 1:1 (green) and 1:2 (red). ( d ) Map of significant chemical shifts induced in cPARP3 1–169 by DNA duplex harbouring a 5′-phosphorylated nick ( > 0.1 p.p.m) or 10-bp 3′-overhang with a recessed 5′-phosphorylated terminus ( > 0.04), surface modelled using CS-Rosetta 41 . Residues with a significant chemical shift in the presence of either a nick (blue) or 3′-overhang (green) or both (red) are indicated. ( e ) Electrostatic surface of modelled cPARP31–169 with nicked DNA. ( f ) Model of cPARP3 1–169 with nicked DNA, depicting residues with significant chemical shifts as above. ( g ) Model of cPARP3 1–169 with nicked DNA lacking the strand located upstream (5′) of the nick (that is, harbouring a DSB with 10-bp 3′-overhang). Residues exhibiting a significant chemical shift are indicated as above.

    Journal: Nature Communications

    Article Title: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2

    doi: 10.1038/ncomms12404

    Figure Lengend Snippet: Mechanism of DNA break binding by the cPARP3 WGR domain. ( a ) Schematic representation of PARP3 domains with the human (top) and chicken (bottom) amino acid positions indicated. The WGR domain is shown in expanded format below showing the HSQC perturbed residues of chicken PARP3 (bottom) and the equivalent residues in human PARP3 (top). ( b ) Overlay of 1 H– 15 N HSQC NMR full spectra for cPARP3 1–169 in the absence (blue) or presence (red) of oligodeoxyribonucleotide duplex harbouring a 5′-phosphorylated nick (protein:DNA ratios of 1:0 and 1:2, respectively). ( c ) Expanded view of the small boxed region shown in b demonstrating the chemical shifts induced in cPARP3 1–169 by different concentrations of nicked DNA. Protein:DNA ratios were 1:0 (that is, no DNA; blue), 5:1 (magenta), 1:1 (green) and 1:2 (red). ( d ) Map of significant chemical shifts induced in cPARP3 1–169 by DNA duplex harbouring a 5′-phosphorylated nick ( > 0.1 p.p.m) or 10-bp 3′-overhang with a recessed 5′-phosphorylated terminus ( > 0.04), surface modelled using CS-Rosetta 41 . Residues with a significant chemical shift in the presence of either a nick (blue) or 3′-overhang (green) or both (red) are indicated. ( e ) Electrostatic surface of modelled cPARP31–169 with nicked DNA. ( f ) Model of cPARP3 1–169 with nicked DNA, depicting residues with significant chemical shifts as above. ( g ) Model of cPARP3 1–169 with nicked DNA lacking the strand located upstream (5′) of the nick (that is, harbouring a DSB with 10-bp 3′-overhang). Residues exhibiting a significant chemical shift are indicated as above.

    Article Snippet: A synthetic codon-optimized gene for expression in Escherichia coli, encoding full-length Gallus gallus PARP3 (chicken PARP3; cPARP3) was purchased from GenScript (Piscataway, NJ, USA).

    Techniques: Binding Assay, Nuclear Magnetic Resonance

    PARP3 promotes chromosomal SSBR and is stimulated by SSBs with canonical termini. ( a ) Wild-type (WT) DT40 cells, PARP3 −/− (PARP3 KO) DT40 cells, and PARP3 −/− DT40 cells stably transfected with either empty vector (vector) or vector encoding human recombinant PARP3 (hPARP3) were treated with the indicated doses of γ-rays and survival calculated in clonogenic assays. Data are the mean (±s.e.m.) of three independent experiments. Where not visible, error bars are smaller than the symbols. ( b ) WT, PARP1 −/− , or PARP3 −/− DT40 cells were treated on ice with γ-rays (20 Gy) and incubated for the indicated times to allow repair. DNA strand breaks were quantified (tail moment) by alkaline comet assays. Data are the average tail moment of > 50 cells per sample and are the mean of three independent experiments (±s.e.m.). ( c ) WT, PARP3 −/− , or derivatives of PARP3 −/− DT40 cells complemented with empty vector or hPARP3 were treated on ice with γ-rays (20 Gy) and incubated for the indicated times to allow repair. DNA strand breaks were quantified as above. ( d ) WT, KU70 −/− , or XRCC3 −/− DT40 cells were treated on ice with γ-rays (20 Gy) and incubated for the indicated times to allow repair. DNA strand breaks were quantified as above. ANOVA was employed to compare mutant DT40 for significant differences with WT (** P

    Journal: Nature Communications

    Article Title: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2

    doi: 10.1038/ncomms12404

    Figure Lengend Snippet: PARP3 promotes chromosomal SSBR and is stimulated by SSBs with canonical termini. ( a ) Wild-type (WT) DT40 cells, PARP3 −/− (PARP3 KO) DT40 cells, and PARP3 −/− DT40 cells stably transfected with either empty vector (vector) or vector encoding human recombinant PARP3 (hPARP3) were treated with the indicated doses of γ-rays and survival calculated in clonogenic assays. Data are the mean (±s.e.m.) of three independent experiments. Where not visible, error bars are smaller than the symbols. ( b ) WT, PARP1 −/− , or PARP3 −/− DT40 cells were treated on ice with γ-rays (20 Gy) and incubated for the indicated times to allow repair. DNA strand breaks were quantified (tail moment) by alkaline comet assays. Data are the average tail moment of > 50 cells per sample and are the mean of three independent experiments (±s.e.m.). ( c ) WT, PARP3 −/− , or derivatives of PARP3 −/− DT40 cells complemented with empty vector or hPARP3 were treated on ice with γ-rays (20 Gy) and incubated for the indicated times to allow repair. DNA strand breaks were quantified as above. ( d ) WT, KU70 −/− , or XRCC3 −/− DT40 cells were treated on ice with γ-rays (20 Gy) and incubated for the indicated times to allow repair. DNA strand breaks were quantified as above. ANOVA was employed to compare mutant DT40 for significant differences with WT (** P

    Article Snippet: A synthetic codon-optimized gene for expression in Escherichia coli, encoding full-length Gallus gallus PARP3 (chicken PARP3; cPARP3) was purchased from GenScript (Piscataway, NJ, USA).

    Techniques: Stable Transfection, Transfection, Plasmid Preparation, Recombinant, Incubation, Mutagenesis

    The PARP3 DNA-binding interface is required for PARP3 stimulation and accumulation at chromosome DNA damage. ( a ) Wild-type or the indicated mutant full-length cPARP3 (300 nM) was incubated for 20 min at room temp with biotin-NAD + (12.5 μM) and 200 nM of oligonucleotide duplex harbouring either a 5′-phosphorylated nick or 5′-phosphorylated DSB with 3′-overhang. Reaction products were separated by SDS–PAGE, blotted, and detected with streptavidin-HRP. Autoribosylated cPARP3 was quantified and plotted relative to that generated in reactions containing nicked duplex and wild type cPARP3. Data are the mean (±s.e.m.) from three independent experiments. ( b ) Time-course of wild-type or mutant cPARP3 incubated from 0 to 30 min in the same conditions as above. ( c ) Recombinant wild-type or mutant cPARP3 (0–0.8 μM) was incubated with a 3′-fluorescein isothiocyanate (FITC)-labeled oligonucleotide duplex harbouring a 5′-phosphorylated nick (100 nM), and protein-DNA complexes detected by EMSA. ( d ) Recruitment of wild-type and mutant human PARP3-GFP to sites of UVA-laser DNA damage in human U2-OS cells. (left) Representative images of WT and mutant PARP3-GFP before treatment (Unt) and 1 min after laser damage. (top right) Quantification of GFP accumulation at sites of laser damage (% increase over initial level). Data are the mean (±s.e.m.) of 25 or more cells per sample. The hPARP3 WGR mutations were Y83A, W101L and R103N and H384A/E514A in the catalytic domain (denoted ‘CM'). ( e , top) PARP3 −/− DT40 cells stably transfected with either empty vector (vector) or vector encoding wild-type hPARP3 (WT) or the mutant derivatives Y83A, W101L and R103N were treated on ice with γ-rays (20 Gy) and incubated for the indicated times to allow repair. DNA strand breaks were quantified (tail moment) by alkaline comet assays. The inset is a western blot showing the expression level of wild type and mutant hPARP3 in PARP3 −/− DT40 cells. (bottom) The above DT40 cell lines were treated with the indicated doses of γ-rays and survival quantified in clonogenic assays. Data are the mean (±s.e.m.) of three independent experiments. Where not visible, error bars are smaller than the symbols. HRP, horseradish peroxidase.

    Journal: Nature Communications

    Article Title: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2

    doi: 10.1038/ncomms12404

    Figure Lengend Snippet: The PARP3 DNA-binding interface is required for PARP3 stimulation and accumulation at chromosome DNA damage. ( a ) Wild-type or the indicated mutant full-length cPARP3 (300 nM) was incubated for 20 min at room temp with biotin-NAD + (12.5 μM) and 200 nM of oligonucleotide duplex harbouring either a 5′-phosphorylated nick or 5′-phosphorylated DSB with 3′-overhang. Reaction products were separated by SDS–PAGE, blotted, and detected with streptavidin-HRP. Autoribosylated cPARP3 was quantified and plotted relative to that generated in reactions containing nicked duplex and wild type cPARP3. Data are the mean (±s.e.m.) from three independent experiments. ( b ) Time-course of wild-type or mutant cPARP3 incubated from 0 to 30 min in the same conditions as above. ( c ) Recombinant wild-type or mutant cPARP3 (0–0.8 μM) was incubated with a 3′-fluorescein isothiocyanate (FITC)-labeled oligonucleotide duplex harbouring a 5′-phosphorylated nick (100 nM), and protein-DNA complexes detected by EMSA. ( d ) Recruitment of wild-type and mutant human PARP3-GFP to sites of UVA-laser DNA damage in human U2-OS cells. (left) Representative images of WT and mutant PARP3-GFP before treatment (Unt) and 1 min after laser damage. (top right) Quantification of GFP accumulation at sites of laser damage (% increase over initial level). Data are the mean (±s.e.m.) of 25 or more cells per sample. The hPARP3 WGR mutations were Y83A, W101L and R103N and H384A/E514A in the catalytic domain (denoted ‘CM'). ( e , top) PARP3 −/− DT40 cells stably transfected with either empty vector (vector) or vector encoding wild-type hPARP3 (WT) or the mutant derivatives Y83A, W101L and R103N were treated on ice with γ-rays (20 Gy) and incubated for the indicated times to allow repair. DNA strand breaks were quantified (tail moment) by alkaline comet assays. The inset is a western blot showing the expression level of wild type and mutant hPARP3 in PARP3 −/− DT40 cells. (bottom) The above DT40 cell lines were treated with the indicated doses of γ-rays and survival quantified in clonogenic assays. Data are the mean (±s.e.m.) of three independent experiments. Where not visible, error bars are smaller than the symbols. HRP, horseradish peroxidase.

    Article Snippet: A synthetic codon-optimized gene for expression in Escherichia coli, encoding full-length Gallus gallus PARP3 (chicken PARP3; cPARP3) was purchased from GenScript (Piscataway, NJ, USA).

    Techniques: Binding Assay, Mutagenesis, Incubation, TNKS1 Histone Ribosylation Assay, SDS Page, Generated, Recombinant, Labeling, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Expressing

    PARP3 monoribosylates H2B in damaged chromatin. ( a , left) 10μg of the chicken chromatin employed in these experiments was fractionated by SDS–PAGE and stained with Coomassie blue. (right) One microgram of soluble MNase-treated chicken chromatin or 50-mer oligonucleotide duplex (200 nM) harbouring a nick with 3′-P/5′-OH termini was mock-treated (0) or treated with 1, 0.5 or 0.25 U T4 PNK to restore 3′-OH/5′-P termini. These DNA substrates were then incubated with 100 nM hPARP3 and 12.5 μM biotin-NAD + for 30 min and biotinylated products separated by 15% SDS–PAGE and detected with streptavidin-HRP. ( b ) 1 μg chicken chromatin or the indicated recombinant histone was incubated with 100 nM hPARP3 in the presence of 300 nM 32 P-NAD + or 12.5 μM biotin-NAD and oligonucleotide harbouring either a DSB (middle) or SSB (right) and the reaction products fractionated by 15% SDS–PAGE and detected by autoradiography or streptavidin-HRP. (left) An aliquot of the chicken chromatin and recombinant histones was fractionated by SDS–PAGE and stained with Coomassie blue. ( c , left) Aliquots of recombinant histone standards were fractionated separately or together as an octamer on triton-acid urea gels and analysed by staining with Coomassie blue. (right) The products of the PARP3 ribosylation reactions conducted in b were fractionated on triton-acid urea gels and analysed by autoradiography. HRP, horseradish peroxidase.

    Journal: Nature Communications

    Article Title: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2

    doi: 10.1038/ncomms12404

    Figure Lengend Snippet: PARP3 monoribosylates H2B in damaged chromatin. ( a , left) 10μg of the chicken chromatin employed in these experiments was fractionated by SDS–PAGE and stained with Coomassie blue. (right) One microgram of soluble MNase-treated chicken chromatin or 50-mer oligonucleotide duplex (200 nM) harbouring a nick with 3′-P/5′-OH termini was mock-treated (0) or treated with 1, 0.5 or 0.25 U T4 PNK to restore 3′-OH/5′-P termini. These DNA substrates were then incubated with 100 nM hPARP3 and 12.5 μM biotin-NAD + for 30 min and biotinylated products separated by 15% SDS–PAGE and detected with streptavidin-HRP. ( b ) 1 μg chicken chromatin or the indicated recombinant histone was incubated with 100 nM hPARP3 in the presence of 300 nM 32 P-NAD + or 12.5 μM biotin-NAD and oligonucleotide harbouring either a DSB (middle) or SSB (right) and the reaction products fractionated by 15% SDS–PAGE and detected by autoradiography or streptavidin-HRP. (left) An aliquot of the chicken chromatin and recombinant histones was fractionated by SDS–PAGE and stained with Coomassie blue. ( c , left) Aliquots of recombinant histone standards were fractionated separately or together as an octamer on triton-acid urea gels and analysed by staining with Coomassie blue. (right) The products of the PARP3 ribosylation reactions conducted in b were fractionated on triton-acid urea gels and analysed by autoradiography. HRP, horseradish peroxidase.

    Article Snippet: A synthetic codon-optimized gene for expression in Escherichia coli, encoding full-length Gallus gallus PARP3 (chicken PARP3; cPARP3) was purchased from GenScript (Piscataway, NJ, USA).

    Techniques: SDS Page, Staining, Incubation, TNKS1 Histone Ribosylation Assay, Recombinant, Autoradiography

    PARP3 binds nicked nucleosomes and ribosylates H2B E2 . ( a ) ADP-ribosylation of E2 in recombinant H2B by hPARP3. MS/MS fragmentation profile of doubly charged PE(+15)PAKSAPAPK (theoretical: 554.3115 m/z ; observed: 554.3113 m/z ; 0.4 p.p.m. mass error), indicating the position of the NH (+15.0109 Da) moiety on glutamate resulting from hydroxylamine derivatization of ADP-ribose. Preferred fragmentation N-terminal to prolines is observed, as expected 43 . ( b ) Mutation of E2 reduces ribosylation of H2B by hPARP3. The products of ribosylation reactions containing 200 nM hPARP3, 12.5 μM biotin-NAD + , 100 nM nicked oligonucleotide duplex (50 bp), and 5 μM of wild-type or mutant recombinant H2B were separated on 15% SDS–PAGE gels and detected by autoradiaography. ( c ) PARP3 binds to nicked mononuclesomes. (left) Reconstituted mononucleosomes were assembled on intact or nicked DNA (Widom positioning sequence 601.2) and nucleosome quality assessed by native gel electrophoresis. (right) representative images of negative stained intact (top) or nicked (bottom) nucleosomes incubated with His-tagged cPARP3, and with the His-tagged protein detected by nanogold Ni-NTA. Scale bars, 50nm. ( d ) PARP3 ribosylates H2B E2 in reconstituted nicked mononucleosomes. Hundred nanomolar of intact or nicked 601.2 DNA, present either as naked duplex or within reconstituted nucleosomes containing wild-type or mutant H2B, was incubated with 100 nM hPARP1 (lanes 1–6) or hPARP3 (lanes 7–12) and either 12.5 μM biotin-NAD + (hPARP3) or 1.5 μM biotin-NAD + (hPARP1, to encourage shorter chain modifications). MS/MS, tandem mass spectrometry.

    Journal: Nature Communications

    Article Title: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2

    doi: 10.1038/ncomms12404

    Figure Lengend Snippet: PARP3 binds nicked nucleosomes and ribosylates H2B E2 . ( a ) ADP-ribosylation of E2 in recombinant H2B by hPARP3. MS/MS fragmentation profile of doubly charged PE(+15)PAKSAPAPK (theoretical: 554.3115 m/z ; observed: 554.3113 m/z ; 0.4 p.p.m. mass error), indicating the position of the NH (+15.0109 Da) moiety on glutamate resulting from hydroxylamine derivatization of ADP-ribose. Preferred fragmentation N-terminal to prolines is observed, as expected 43 . ( b ) Mutation of E2 reduces ribosylation of H2B by hPARP3. The products of ribosylation reactions containing 200 nM hPARP3, 12.5 μM biotin-NAD + , 100 nM nicked oligonucleotide duplex (50 bp), and 5 μM of wild-type or mutant recombinant H2B were separated on 15% SDS–PAGE gels and detected by autoradiaography. ( c ) PARP3 binds to nicked mononuclesomes. (left) Reconstituted mononucleosomes were assembled on intact or nicked DNA (Widom positioning sequence 601.2) and nucleosome quality assessed by native gel electrophoresis. (right) representative images of negative stained intact (top) or nicked (bottom) nucleosomes incubated with His-tagged cPARP3, and with the His-tagged protein detected by nanogold Ni-NTA. Scale bars, 50nm. ( d ) PARP3 ribosylates H2B E2 in reconstituted nicked mononucleosomes. Hundred nanomolar of intact or nicked 601.2 DNA, present either as naked duplex or within reconstituted nucleosomes containing wild-type or mutant H2B, was incubated with 100 nM hPARP1 (lanes 1–6) or hPARP3 (lanes 7–12) and either 12.5 μM biotin-NAD + (hPARP3) or 1.5 μM biotin-NAD + (hPARP1, to encourage shorter chain modifications). MS/MS, tandem mass spectrometry.

    Article Snippet: A synthetic codon-optimized gene for expression in Escherichia coli, encoding full-length Gallus gallus PARP3 (chicken PARP3; cPARP3) was purchased from GenScript (Piscataway, NJ, USA).

    Techniques: Recombinant, Mass Spectrometry, Mutagenesis, TNKS1 Histone Ribosylation Assay, SDS Page, Sequencing, Nucleic Acid Electrophoresis, Staining, Incubation

    CARM1 represses γ-globin expression. A , Western blot analysis of indicated protein levels in cell lysates from the scramble control (Scr), CARM1-KD1, and CARM1-KD2 Lys-562 cells. GAPDH and histone H4 were used as loading controls. Blots are representative of three independent experiments. B and C , quantitative real-time PCR analysis of CARM1 ( B ) and γ-globin ( C ) mRNA level in Scr, CARM1-KD1, and CARM1-KD2 Lys-562 cells normalized to β-actin mRNA. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. **, p

    Journal: The Journal of Biological Chemistry

    Article Title: CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells

    doi: 10.1074/jbc.RA118.004028

    Figure Lengend Snippet: CARM1 represses γ-globin expression. A , Western blot analysis of indicated protein levels in cell lysates from the scramble control (Scr), CARM1-KD1, and CARM1-KD2 Lys-562 cells. GAPDH and histone H4 were used as loading controls. Blots are representative of three independent experiments. B and C , quantitative real-time PCR analysis of CARM1 ( B ) and γ-globin ( C ) mRNA level in Scr, CARM1-KD1, and CARM1-KD2 Lys-562 cells normalized to β-actin mRNA. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. **, p

    Article Snippet: The following antibodies were used for Western blotting: FLAG (Sigma-Aldrich), PRMT5 (Sigma-Aldrich), CARM1 (Cell Signaling Technology), GAPDH (MBL International), H4R3me2s (Abcam), H4R3me2a (Active Motif), H3R17me2a (Abcam), histone H4 (GenScript), and histone H3 (GenScript).

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

    CARM1 methylates PRMT5. A , SDS-PAGE analysis of purified recombinant PRMT5 (rPRMT5) from E. coli as the substrate for following in vitro methylation assays. B , SDS-PAGE analysis of purified recombinant GST-PRMT1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-PRMT1 or GST control ( right ). C , SDS-PAGE analysis of purified recombinant GST-CARM1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H3R17me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-CARM1 or GST control ( right ). A gray asterisk denotes the positive 3 H-labeled PRMT5 band. D , SDS-PAGE analysis of IgG control ( top left ) and of FLAG-PRMT5 immunoprecipitated from Lys-562 cells overexpressing FLAG-tagged PRMT5. SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2s antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with FLAG-PRMT5 or IgG control ( right ). Black asterisks denote the fusion proteins in assays.

    Journal: The Journal of Biological Chemistry

    Article Title: CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells

    doi: 10.1074/jbc.RA118.004028

    Figure Lengend Snippet: CARM1 methylates PRMT5. A , SDS-PAGE analysis of purified recombinant PRMT5 (rPRMT5) from E. coli as the substrate for following in vitro methylation assays. B , SDS-PAGE analysis of purified recombinant GST-PRMT1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-PRMT1 or GST control ( right ). C , SDS-PAGE analysis of purified recombinant GST-CARM1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H3R17me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-CARM1 or GST control ( right ). A gray asterisk denotes the positive 3 H-labeled PRMT5 band. D , SDS-PAGE analysis of IgG control ( top left ) and of FLAG-PRMT5 immunoprecipitated from Lys-562 cells overexpressing FLAG-tagged PRMT5. SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2s antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with FLAG-PRMT5 or IgG control ( right ). Black asterisks denote the fusion proteins in assays.

    Article Snippet: The following antibodies were used for Western blotting: FLAG (Sigma-Aldrich), PRMT5 (Sigma-Aldrich), CARM1 (Cell Signaling Technology), GAPDH (MBL International), H4R3me2s (Abcam), H4R3me2a (Active Motif), H3R17me2a (Abcam), histone H4 (GenScript), and histone H3 (GenScript).

    Techniques: SDS Page, Purification, Recombinant, In Vitro, Methylation, Staining, Western Blot, Labeling, Immunoprecipitation

    Methylation of PRMT5 at Arg-505 is essential for its methyltransferase activity. A , Western blot analysis of extracts from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using FLAG and PRMT5 antibodies. GAPDH was used as a loading control. Blots are representative of three independent experiments. B, quantitative real-time PCR analysis of PRMT5 mRNA normalized to β-actin in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. C , Western blot analysis of extracted histones from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using H4R3me2s antibody. Histone H4 was used as a loading control. Blots are representative of three independent experiments. D , ChIP analysis of H4R3me2s enrichment at the γ-promoter in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells

    doi: 10.1074/jbc.RA118.004028

    Figure Lengend Snippet: Methylation of PRMT5 at Arg-505 is essential for its methyltransferase activity. A , Western blot analysis of extracts from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using FLAG and PRMT5 antibodies. GAPDH was used as a loading control. Blots are representative of three independent experiments. B, quantitative real-time PCR analysis of PRMT5 mRNA normalized to β-actin in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. C , Western blot analysis of extracted histones from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using H4R3me2s antibody. Histone H4 was used as a loading control. Blots are representative of three independent experiments. D , ChIP analysis of H4R3me2s enrichment at the γ-promoter in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. *, p

    Article Snippet: The following antibodies were used for Western blotting: FLAG (Sigma-Aldrich), PRMT5 (Sigma-Aldrich), CARM1 (Cell Signaling Technology), GAPDH (MBL International), H4R3me2s (Abcam), H4R3me2a (Active Motif), H3R17me2a (Abcam), histone H4 (GenScript), and histone H3 (GenScript).

    Techniques: Methylation, Activity Assay, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Two Tailed Test