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  • 99
    New England Biolabs t7 rnap
     are shown above. VSR10 is made of a double-stranded DNA (black) annealed to a 10-nt RNA primer (red). The RNA is labelled at its 5′ end with [γ- 32 P]ATP. The arrow indicates the nucleotide incorporation site. ( A ) UTP and CTP incorporation rates at different nucleotide concentrations, obtained at pH 7.9 and 6 mM MgCl 2  (black circles), and at pH 6.75 and 1.5 mM MgCl 2  (grey circles). ( B ) Pre-steady-state kinetics of CTP incorporation on VSR10 by T7 RNAP, at pH 6.75 (Bis-Tris buffer) and 1.5 mM MgCl 2 . In the left panel, continuous lines represent the best fit of the data to the single-exponential equation, obtained at different nucleotide concentrations. The right panel shows the nucleotide concentration dependence of CTP incorporation. The continuous line represents the best fit of the  k obs  data to the Michaelis-Menten equation. The obtained polymerization rates ( k pol ) and apparent equilibrium dissociation constant ( K d ) of CTP were 4.98 ± 0.71 s −1  and 2.23 ± 0.61 mM, respectively. Results were obtained from three independent experiments.
    T7 Rnap, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Santa Cruz Biotechnology rnap ii
     are shown above. VSR10 is made of a double-stranded DNA (black) annealed to a 10-nt RNA primer (red). The RNA is labelled at its 5′ end with [γ- 32 P]ATP. The arrow indicates the nucleotide incorporation site. ( A ) UTP and CTP incorporation rates at different nucleotide concentrations, obtained at pH 7.9 and 6 mM MgCl 2  (black circles), and at pH 6.75 and 1.5 mM MgCl 2  (grey circles). ( B ) Pre-steady-state kinetics of CTP incorporation on VSR10 by T7 RNAP, at pH 6.75 (Bis-Tris buffer) and 1.5 mM MgCl 2 . In the left panel, continuous lines represent the best fit of the data to the single-exponential equation, obtained at different nucleotide concentrations. The right panel shows the nucleotide concentration dependence of CTP incorporation. The continuous line represents the best fit of the  k obs  data to the Michaelis-Menten equation. The obtained polymerization rates ( k pol ) and apparent equilibrium dissociation constant ( K d ) of CTP were 4.98 ± 0.71 s −1  and 2.23 ± 0.61 mM, respectively. Results were obtained from three independent experiments.
    Rnap Ii, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Covance rnap ii
    <t>RNAP</t> II reverse the inhibitory effect of DNA damage on 3′ processing. ( A ) Activation of cleavage by the addition of GST-CTD. <t>NEs</t> active (0-h UV treatment) and inactive (5-h UV treatment) for 3′ cleavage were preincubated with no addition (lanes 1,2,5,6 ) or with increasing amounts (100 and 200 ng) of recombinant GST-CTD (lanes 3,4,7,8 ). The cleavage reactions were performed in the absence (lanes 1,3 – 5,7,8 ) or presence (lanes 2,6 ) of creatine phosphate. Positions of pre-mRNA and the 5′ and 3′ cleavage products are indicated. ( B ) Activation of cleavage by the addition of RNAP IIO. NEs inactive (5-h UV treatment) for 3′ cleavage were preincubated with no addition (lane 1 ) or with increasing amounts (25, 50, and 100 ng) of purified RNAP IIO (lanes 2 – 4 ). Positions of pre-mRNA and the 5′ and 3′ cleavage products are indicated.
    Rnap Ii, supplied by Covance, 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
    BioLegend rnap ii
    Protein interaction network of the <t>RNAP</t> <t>II/U1</t> snRNP machinery. The interaction network of the RNAP II/U1 snRNP machinery was built using the STRING database (confidence > 0.7 based on experimental data) together with manual changes as described in the main text. Distinct complexes are labeled in the black boxes. ALS/SMA-causative proteins are marked in bright red.
    Rnap Ii, supplied by BioLegend, 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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    Image Search Results


     are shown above. VSR10 is made of a double-stranded DNA (black) annealed to a 10-nt RNA primer (red). The RNA is labelled at its 5′ end with [γ- 32 P]ATP. The arrow indicates the nucleotide incorporation site. ( A ) UTP and CTP incorporation rates at different nucleotide concentrations, obtained at pH 7.9 and 6 mM MgCl 2  (black circles), and at pH 6.75 and 1.5 mM MgCl 2  (grey circles). ( B ) Pre-steady-state kinetics of CTP incorporation on VSR10 by T7 RNAP, at pH 6.75 (Bis-Tris buffer) and 1.5 mM MgCl 2 . In the left panel, continuous lines represent the best fit of the data to the single-exponential equation, obtained at different nucleotide concentrations. The right panel shows the nucleotide concentration dependence of CTP incorporation. The continuous line represents the best fit of the  k obs  data to the Michaelis-Menten equation. The obtained polymerization rates ( k pol ) and apparent equilibrium dissociation constant ( K d ) of CTP were 4.98 ± 0.71 s −1  and 2.23 ± 0.61 mM, respectively. Results were obtained from three independent experiments.

    Journal: Scientific Reports

    Article Title: Transcriptional inaccuracy threshold attenuates differences in RNA-dependent DNA synthesis fidelity between retroviral reverse transcriptases

    doi: 10.1038/s41598-017-18974-8

    Figure Lengend Snippet: are shown above. VSR10 is made of a double-stranded DNA (black) annealed to a 10-nt RNA primer (red). The RNA is labelled at its 5′ end with [γ- 32 P]ATP. The arrow indicates the nucleotide incorporation site. ( A ) UTP and CTP incorporation rates at different nucleotide concentrations, obtained at pH 7.9 and 6 mM MgCl 2 (black circles), and at pH 6.75 and 1.5 mM MgCl 2 (grey circles). ( B ) Pre-steady-state kinetics of CTP incorporation on VSR10 by T7 RNAP, at pH 6.75 (Bis-Tris buffer) and 1.5 mM MgCl 2 . In the left panel, continuous lines represent the best fit of the data to the single-exponential equation, obtained at different nucleotide concentrations. The right panel shows the nucleotide concentration dependence of CTP incorporation. The continuous line represents the best fit of the k obs data to the Michaelis-Menten equation. The obtained polymerization rates ( k pol ) and apparent equilibrium dissociation constant ( K d ) of CTP were 4.98 ± 0.71 s −1 and 2.23 ± 0.61 mM, respectively. Results were obtained from three independent experiments.

    Article Snippet: Briefly, the dsDNA genome of the M13mp2 bacteriophage containing the promoter for T7 RNAP upstream the lac Zα gene (T7-M13mp2) was digested with FspI (New England Biolabs).

    Techniques: Concentration Assay

    RNAP II reverse the inhibitory effect of DNA damage on 3′ processing. ( A ) Activation of cleavage by the addition of GST-CTD. NEs active (0-h UV treatment) and inactive (5-h UV treatment) for 3′ cleavage were preincubated with no addition (lanes 1,2,5,6 ) or with increasing amounts (100 and 200 ng) of recombinant GST-CTD (lanes 3,4,7,8 ). The cleavage reactions were performed in the absence (lanes 1,3 – 5,7,8 ) or presence (lanes 2,6 ) of creatine phosphate. Positions of pre-mRNA and the 5′ and 3′ cleavage products are indicated. ( B ) Activation of cleavage by the addition of RNAP IIO. NEs inactive (5-h UV treatment) for 3′ cleavage were preincubated with no addition (lane 1 ) or with increasing amounts (25, 50, and 100 ng) of purified RNAP IIO (lanes 2 – 4 ). Positions of pre-mRNA and the 5′ and 3′ cleavage products are indicated.

    Journal: Genes & Development

    Article Title: BRCA1/BARD1 inhibition of mRNA 3? processing involves targeted degradation of RNA polymerase II

    doi: 10.1101/gad.1309505

    Figure Lengend Snippet: RNAP II reverse the inhibitory effect of DNA damage on 3′ processing. ( A ) Activation of cleavage by the addition of GST-CTD. NEs active (0-h UV treatment) and inactive (5-h UV treatment) for 3′ cleavage were preincubated with no addition (lanes 1,2,5,6 ) or with increasing amounts (100 and 200 ng) of recombinant GST-CTD (lanes 3,4,7,8 ). The cleavage reactions were performed in the absence (lanes 1,3 – 5,7,8 ) or presence (lanes 2,6 ) of creatine phosphate. Positions of pre-mRNA and the 5′ and 3′ cleavage products are indicated. ( B ) Activation of cleavage by the addition of RNAP IIO. NEs inactive (5-h UV treatment) for 3′ cleavage were preincubated with no addition (lane 1 ) or with increasing amounts (25, 50, and 100 ng) of purified RNAP IIO (lanes 2 – 4 ). Positions of pre-mRNA and the 5′ and 3′ cleavage products are indicated.

    Article Snippet: Sixty micrograms of the NEs were analyzed by immunoblotting with mAbs targeted against RNAP II (8WG16, Covance, and N-20, Santa Cruz), RNAP IIO (H5; Covance), BRCA1 (C-20, Santa Cruz), BARD1 (H-300, Santa Cruz), and actin (A 2066; Sigma).

    Techniques: Activation Assay, Recombinant, Purification

    RNAP IIO but not RNAP IIA is ubiquitinated by BRCA1/BARD1. ( A ) RNAP IIO was incubated with E1, E2, His-HA-Ub, and a purified heterodimer comprised of truncated BRCA1 and full-length BARD1 (ΔBRCA1/BARD1-wt) as indicated (lanes 1 – 3,5 ). Lane 4 shows a reaction containing the mutant heterodimer ΔBRCA1/BARD1-C61G. Reactions were terminated and proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-RNAP II LS and anti-Ub antibodies in the top and lower panels, respectively. Positions of the RNAP IIO and RNAP IIA forms are indicated on the left , and the polyubiquitinated forms of RNAP IIO and molecular-weight markers are indicated on the right . ( B ) Kinetics of RNAP IIO ubiquitination by ΔBRCA1/BARD1-wt. Ubiquitination reactions were performed and analyzed as in A , except that the incubation times were as indicated. ( C ) Ubiquitination of RNAP IIO with a heterodimeric complex comprised of full-length BRCA1 and BARD1. Reactions were performed and analyzed as in A . ( D ) ΔBRCA1/BARD1-wt stimulates polyubiquitination of RNAP IIO but not RNAP IIA. Ubiquitination reactions were performed as above except in presence of either RNAP IIO or RNAP IIA. ( E ) ΔBRCA1/BARD1 does not stimulate ubiquitination of either GST-CTD or phosphorylated GST-CTD. Ubiquitination reactions were done with nonphosphorylated (lanes 1 – 3 ) or in vitro phosphorylated (lanes 4 – 6 ) GST-CTD as in panel A . Positions of the nonphosphorylated and phosphorylated GST-CTD proteins are indicated on the left . ( F ) Concentration and purity of RNAP IIO and RNAP IIA (lanes 1,2 ; only the largest subunits are shown), GST-CTD and pGST-CTD (lanes 3,4 ), and the heterodimeric complexes comprised of full-length BARD1 and truncated wild-type and C61G BRCA1 (lanes 5,6 ) were monitored by silver or Coomassie blue staining following SDS-PAGE. Positions of the three largest RNAP II subunits (IIa, IIo, and IIc) are indicated on the left , and positions of molecular-weight markers are indicated on the right .( G ) Schematic diagrams of BRCA1 (1863 amino acids) and BARD1 (777 amino acids) showing the N-terminal RING domains (BRCA1 23–76, BARD1 49–100), the ankyrin repeats (BARD1 427–525), and C-terminal BRCT domains.

    Journal: Genes & Development

    Article Title: BRCA1/BARD1 inhibition of mRNA 3? processing involves targeted degradation of RNA polymerase II

    doi: 10.1101/gad.1309505

    Figure Lengend Snippet: RNAP IIO but not RNAP IIA is ubiquitinated by BRCA1/BARD1. ( A ) RNAP IIO was incubated with E1, E2, His-HA-Ub, and a purified heterodimer comprised of truncated BRCA1 and full-length BARD1 (ΔBRCA1/BARD1-wt) as indicated (lanes 1 – 3,5 ). Lane 4 shows a reaction containing the mutant heterodimer ΔBRCA1/BARD1-C61G. Reactions were terminated and proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-RNAP II LS and anti-Ub antibodies in the top and lower panels, respectively. Positions of the RNAP IIO and RNAP IIA forms are indicated on the left , and the polyubiquitinated forms of RNAP IIO and molecular-weight markers are indicated on the right . ( B ) Kinetics of RNAP IIO ubiquitination by ΔBRCA1/BARD1-wt. Ubiquitination reactions were performed and analyzed as in A , except that the incubation times were as indicated. ( C ) Ubiquitination of RNAP IIO with a heterodimeric complex comprised of full-length BRCA1 and BARD1. Reactions were performed and analyzed as in A . ( D ) ΔBRCA1/BARD1-wt stimulates polyubiquitination of RNAP IIO but not RNAP IIA. Ubiquitination reactions were performed as above except in presence of either RNAP IIO or RNAP IIA. ( E ) ΔBRCA1/BARD1 does not stimulate ubiquitination of either GST-CTD or phosphorylated GST-CTD. Ubiquitination reactions were done with nonphosphorylated (lanes 1 – 3 ) or in vitro phosphorylated (lanes 4 – 6 ) GST-CTD as in panel A . Positions of the nonphosphorylated and phosphorylated GST-CTD proteins are indicated on the left . ( F ) Concentration and purity of RNAP IIO and RNAP IIA (lanes 1,2 ; only the largest subunits are shown), GST-CTD and pGST-CTD (lanes 3,4 ), and the heterodimeric complexes comprised of full-length BARD1 and truncated wild-type and C61G BRCA1 (lanes 5,6 ) were monitored by silver or Coomassie blue staining following SDS-PAGE. Positions of the three largest RNAP II subunits (IIa, IIo, and IIc) are indicated on the left , and positions of molecular-weight markers are indicated on the right .( G ) Schematic diagrams of BRCA1 (1863 amino acids) and BARD1 (777 amino acids) showing the N-terminal RING domains (BRCA1 23–76, BARD1 49–100), the ankyrin repeats (BARD1 427–525), and C-terminal BRCT domains.

    Article Snippet: Sixty micrograms of the NEs were analyzed by immunoblotting with mAbs targeted against RNAP II (8WG16, Covance, and N-20, Santa Cruz), RNAP IIO (H5; Covance), BRCA1 (C-20, Santa Cruz), BARD1 (H-300, Santa Cruz), and actin (A 2066; Sigma).

    Techniques: Incubation, Purification, Mutagenesis, SDS Page, Molecular Weight, In Vitro, Concentration Assay, Staining

    siRNA knockdown of both BRCA1/BARD1 expression abolishes UV-induced degradation of RNAP II. ( A ) Protein levels of BRCA1, BARD1, actin, and RNAP II in NEs prepared from cells subjected to control (cont), BRCA1, BARD1, and BRCA1/BARD1 siRNA and UV irradiation. Nonirradiated and UV-irradiated samples were prepared 48 h after the addition of the siRNAs. Irradiated cells were allowed to recover 2 h after exposure to UV doses of 10 Jm –2 . Panels depict blots using antibodies against BRCA1, BARD1, RNAP IIO (H5), RNAP II (N-20), and actin. Protein concentrations were equalized by immunostaining with antibodies against actin. The actin and the RNAP II blots correspond to the same gel. The positions of each protein are indicated in the corresponding panel. ( B ) An inhibitor of the proteasome prevents UV-induced degradation but not ubiquitination of RNAP II in siRNA-treated cells. siRNA-treated cells were irradiated with UV and allowed to recover in the presence of the proteasomal inhibitor MG132 for 2 h. BRCA1, BARD1, RNAP II, RNAP IIO, and actin protein levels in NEs from these cells were analyzed by Western blot as above.

    Journal: Genes & Development

    Article Title: BRCA1/BARD1 inhibition of mRNA 3? processing involves targeted degradation of RNA polymerase II

    doi: 10.1101/gad.1309505

    Figure Lengend Snippet: siRNA knockdown of both BRCA1/BARD1 expression abolishes UV-induced degradation of RNAP II. ( A ) Protein levels of BRCA1, BARD1, actin, and RNAP II in NEs prepared from cells subjected to control (cont), BRCA1, BARD1, and BRCA1/BARD1 siRNA and UV irradiation. Nonirradiated and UV-irradiated samples were prepared 48 h after the addition of the siRNAs. Irradiated cells were allowed to recover 2 h after exposure to UV doses of 10 Jm –2 . Panels depict blots using antibodies against BRCA1, BARD1, RNAP IIO (H5), RNAP II (N-20), and actin. Protein concentrations were equalized by immunostaining with antibodies against actin. The actin and the RNAP II blots correspond to the same gel. The positions of each protein are indicated in the corresponding panel. ( B ) An inhibitor of the proteasome prevents UV-induced degradation but not ubiquitination of RNAP II in siRNA-treated cells. siRNA-treated cells were irradiated with UV and allowed to recover in the presence of the proteasomal inhibitor MG132 for 2 h. BRCA1, BARD1, RNAP II, RNAP IIO, and actin protein levels in NEs from these cells were analyzed by Western blot as above.

    Article Snippet: Sixty micrograms of the NEs were analyzed by immunoblotting with mAbs targeted against RNAP II (8WG16, Covance, and N-20, Santa Cruz), RNAP IIO (H5; Covance), BRCA1 (C-20, Santa Cruz), BARD1 (H-300, Santa Cruz), and actin (A 2066; Sigma).

    Techniques: Expressing, Irradiation, Immunostaining, Western Blot

    Role of protein turnover and RNAP II phosphorylation in UV-induced inhibition of 3′ processing. ( A ) An inhibitor of the proteasome rescues UV-induced inhibition of 3′ cleavage. HeLa cells were treated with UV irradiation and allowed to recover in the presence (lanes 1 – 5 ) or absence (lanes 6 – 10 ) of the proteasomal inhibitor MG132 for the times indicated. NEs prepared from these cells were used in cleavage reactions with an SV40 late ( top ) or adenovirus L3 ( bottom ) pre-mRNA. Positions of pre-mRNA and the 5′ and 3′ cleavage products are indicated. ( B ) The CTD-kinase inhibitor H7 blocks UV-induced inhibition of 3′ cleavage. HeLa cells were treated with UV irradiation and allowed to recover in the absence (lanes 1 – 5 ) or presence (lanes 6 – 10 ) of H7 for the times indicated. Cells were also treated with H7 only (lanes 11 – 14 ). NEs prepared from these cells were used in 3′ cleavage reactions as above. Positions of the SV40 pre-mRNA and 5′ and 3′ cleavage products are indicated. RNAP IIO, RNAP IIA, and actin protein levels in NEs from treated cells were monitored by Western blotting. Proteins were detected by immunoblotting with antibodies against a nonphosphorylated CTD epitope of RNAP II LS (8WG16) and actin.

    Journal: Genes & Development

    Article Title: BRCA1/BARD1 inhibition of mRNA 3? processing involves targeted degradation of RNA polymerase II

    doi: 10.1101/gad.1309505

    Figure Lengend Snippet: Role of protein turnover and RNAP II phosphorylation in UV-induced inhibition of 3′ processing. ( A ) An inhibitor of the proteasome rescues UV-induced inhibition of 3′ cleavage. HeLa cells were treated with UV irradiation and allowed to recover in the presence (lanes 1 – 5 ) or absence (lanes 6 – 10 ) of the proteasomal inhibitor MG132 for the times indicated. NEs prepared from these cells were used in cleavage reactions with an SV40 late ( top ) or adenovirus L3 ( bottom ) pre-mRNA. Positions of pre-mRNA and the 5′ and 3′ cleavage products are indicated. ( B ) The CTD-kinase inhibitor H7 blocks UV-induced inhibition of 3′ cleavage. HeLa cells were treated with UV irradiation and allowed to recover in the absence (lanes 1 – 5 ) or presence (lanes 6 – 10 ) of H7 for the times indicated. Cells were also treated with H7 only (lanes 11 – 14 ). NEs prepared from these cells were used in 3′ cleavage reactions as above. Positions of the SV40 pre-mRNA and 5′ and 3′ cleavage products are indicated. RNAP IIO, RNAP IIA, and actin protein levels in NEs from treated cells were monitored by Western blotting. Proteins were detected by immunoblotting with antibodies against a nonphosphorylated CTD epitope of RNAP II LS (8WG16) and actin.

    Article Snippet: Sixty micrograms of the NEs were analyzed by immunoblotting with mAbs targeted against RNAP II (8WG16, Covance, and N-20, Santa Cruz), RNAP IIO (H5; Covance), BRCA1 (C-20, Santa Cruz), BARD1 (H-300, Santa Cruz), and actin (A 2066; Sigma).

    Techniques: Inhibition, Irradiation, Western Blot

    UV treatment transiently disperses BRCA1/BARD1 nuclear foci, RNAP IIO dots, and cleavage bodies, but not SC35 speckles. Asynchronous MCF7 cells were treated with UV and were fixed in 2% formaldehyde after the UV treatment at the times indicated. Fixed cells were immunostained with a BRCA1 mAb (MS110), BARD1 polyclonal antibody (669D), CstF-64 mAb, RNAP IIO LS mAb (H5), and an SC35 mAb. Immunostaining with monoclonal or polyclonal antibodies was visualized by using a goat anti-mouse or anti-rabbit IgG-fluorescein, respectively.

    Journal: Genes & Development

    Article Title: BRCA1/BARD1 inhibition of mRNA 3? processing involves targeted degradation of RNA polymerase II

    doi: 10.1101/gad.1309505

    Figure Lengend Snippet: UV treatment transiently disperses BRCA1/BARD1 nuclear foci, RNAP IIO dots, and cleavage bodies, but not SC35 speckles. Asynchronous MCF7 cells were treated with UV and were fixed in 2% formaldehyde after the UV treatment at the times indicated. Fixed cells were immunostained with a BRCA1 mAb (MS110), BARD1 polyclonal antibody (669D), CstF-64 mAb, RNAP IIO LS mAb (H5), and an SC35 mAb. Immunostaining with monoclonal or polyclonal antibodies was visualized by using a goat anti-mouse or anti-rabbit IgG-fluorescein, respectively.

    Article Snippet: Sixty micrograms of the NEs were analyzed by immunoblotting with mAbs targeted against RNAP II (8WG16, Covance, and N-20, Santa Cruz), RNAP IIO (H5; Covance), BRCA1 (C-20, Santa Cruz), BARD1 (H-300, Santa Cruz), and actin (A 2066; Sigma).

    Techniques: Immunostaining

    Protein interaction network of the RNAP II/U1 snRNP machinery. The interaction network of the RNAP II/U1 snRNP machinery was built using the STRING database (confidence > 0.7 based on experimental data) together with manual changes as described in the main text. Distinct complexes are labeled in the black boxes. ALS/SMA-causative proteins are marked in bright red.

    Journal: Nucleic Acids Research

    Article Title: The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex

    doi: 10.1093/nar/gky1093

    Figure Lengend Snippet: Protein interaction network of the RNAP II/U1 snRNP machinery. The interaction network of the RNAP II/U1 snRNP machinery was built using the STRING database (confidence > 0.7 based on experimental data) together with manual changes as described in the main text. Distinct complexes are labeled in the black boxes. ALS/SMA-causative proteins are marked in bright red.

    Article Snippet: Consistent with this possibility, GST-SRSF1, which pulls down U1 snRNP also pulls down RNAP II, supporting an association between these two machineries ( ).

    Techniques: Labeling

    ALS protein KOs disrupt association of distinct complexes with the RNAP II/U1 snRNP machinery. Protein complexes that dissociate from the RNAP II/U1 snRNP machinery in FUS, EWSR1, TAF15 or MATR3 KO lines are shown. The Y axis indicates fold change (FC).

    Journal: Nucleic Acids Research

    Article Title: The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex

    doi: 10.1093/nar/gky1093

    Figure Lengend Snippet: ALS protein KOs disrupt association of distinct complexes with the RNAP II/U1 snRNP machinery. Protein complexes that dissociate from the RNAP II/U1 snRNP machinery in FUS, EWSR1, TAF15 or MATR3 KO lines are shown. The Y axis indicates fold change (FC).

    Article Snippet: Consistent with this possibility, GST-SRSF1, which pulls down U1 snRNP also pulls down RNAP II, supporting an association between these two machineries ( ).

    Techniques:

    All four ALS proteins mediate association of the ASC-1 complex with the RNAP II/U1 snRNP machinery. ( A ) IP/Western analysis of ASC-1 components associated with the RNAP II/U1 snRNP machinery in WT or KO lines. ( B ) Three independent replicates of the IP/Westerns shown in (A) were quantitated. The bars indicate the mean values of fold change. Error bars represent standard deviations. * P

    Journal: Nucleic Acids Research

    Article Title: The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex

    doi: 10.1093/nar/gky1093

    Figure Lengend Snippet: All four ALS proteins mediate association of the ASC-1 complex with the RNAP II/U1 snRNP machinery. ( A ) IP/Western analysis of ASC-1 components associated with the RNAP II/U1 snRNP machinery in WT or KO lines. ( B ) Three independent replicates of the IP/Westerns shown in (A) were quantitated. The bars indicate the mean values of fold change. Error bars represent standard deviations. * P

    Article Snippet: Consistent with this possibility, GST-SRSF1, which pulls down U1 snRNP also pulls down RNAP II, supporting an association between these two machineries ( ).

    Techniques: Western Blot

    Top hit proteins dissociated from the RNAP II/U1 snRNP machinery in ALS protein KOs. The top ranked (by fold change) proteins that dissociated from the RNAP II/U1 snRNP machinery in each KO line are shown. The rank, HGNC official symbol, calculated molecular weight (kD), best-known function, and fold change relative to wild type are shown. Functions in splicing (pink), transcription (txn, orange), DNA damage response (green), tRNA splicing ligase complex (purple) and other (black) are indicated.

    Journal: Nucleic Acids Research

    Article Title: The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex

    doi: 10.1093/nar/gky1093

    Figure Lengend Snippet: Top hit proteins dissociated from the RNAP II/U1 snRNP machinery in ALS protein KOs. The top ranked (by fold change) proteins that dissociated from the RNAP II/U1 snRNP machinery in each KO line are shown. The rank, HGNC official symbol, calculated molecular weight (kD), best-known function, and fold change relative to wild type are shown. Functions in splicing (pink), transcription (txn, orange), DNA damage response (green), tRNA splicing ligase complex (purple) and other (black) are indicated.

    Article Snippet: Consistent with this possibility, GST-SRSF1, which pulls down U1 snRNP also pulls down RNAP II, supporting an association between these two machineries ( ).

    Techniques: Molecular Weight

    ALS proteins associate with the RNAP II/U1 snRNP machinery in an RNA-independent manner. IPs were carried out from RNase A-treated or untreated nuclear extract using an RNAP II or a negative control antibody (EIF4A3) followed by westerns with the indicated antibodies.

    Journal: Nucleic Acids Research

    Article Title: The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex

    doi: 10.1093/nar/gky1093

    Figure Lengend Snippet: ALS proteins associate with the RNAP II/U1 snRNP machinery in an RNA-independent manner. IPs were carried out from RNase A-treated or untreated nuclear extract using an RNAP II or a negative control antibody (EIF4A3) followed by westerns with the indicated antibodies.

    Article Snippet: Consistent with this possibility, GST-SRSF1, which pulls down U1 snRNP also pulls down RNAP II, supporting an association between these two machineries ( ).

    Techniques: Negative Control

    Disease-causing mutations in FUS or TRIP4 disrupt interactions of ASC-1 complex components with the RNAP II/U1 snRNP machinery. ( A ) Purified recombinant GST-FUS or GST-DDX39B was added to FUS KO extract, and RNAP II was IP’d followed by Westerns with antibodies to the ASC-1 components. The asterisk indicates degradation products of GST-FUS. Endogenous FUS, GST-FUS, and GST-DDX39B were detected by the FUS antibody, as this antibody recognizes GST-DDX39B due to the GST tag common to both proteins. ( B ) Three independent replicates of the data shown in (A) were quantitated. The colored bars in the graph show the mean values of fold change for the indicated proteins. Error bars represent standard deviations. * P

    Journal: Nucleic Acids Research

    Article Title: The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex

    doi: 10.1093/nar/gky1093

    Figure Lengend Snippet: Disease-causing mutations in FUS or TRIP4 disrupt interactions of ASC-1 complex components with the RNAP II/U1 snRNP machinery. ( A ) Purified recombinant GST-FUS or GST-DDX39B was added to FUS KO extract, and RNAP II was IP’d followed by Westerns with antibodies to the ASC-1 components. The asterisk indicates degradation products of GST-FUS. Endogenous FUS, GST-FUS, and GST-DDX39B were detected by the FUS antibody, as this antibody recognizes GST-DDX39B due to the GST tag common to both proteins. ( B ) Three independent replicates of the data shown in (A) were quantitated. The colored bars in the graph show the mean values of fold change for the indicated proteins. Error bars represent standard deviations. * P

    Article Snippet: Consistent with this possibility, GST-SRSF1, which pulls down U1 snRNP also pulls down RNAP II, supporting an association between these two machineries ( ).

    Techniques: Purification, Recombinant