Structured Review

Thermo Fisher sfpq
<t>BCL9</t> regulates mRNA levels of calcium wave-associated genes through paraspeckle proteins. a Network of BCL9-interacting proteins identified by Co-IP MS. Each node represents a group of BCL9-interacting proteins with functional relationships. Lines between different nodes represent the “weight” of the protein-protein interaction according to String database (colored) or total peptides in Co-IP assay (black). Groups were clustered by k-means unsupervised classification according to the interacting “weight” among different nodes. b Representative IF showing co-localization of BCL9, NONO, and ILF2 in CRC but not in normal colon epithelial cells. Scale bars: 20 µm (top, left), and 2 µm (inset). c , d <t>SFPQ</t> binding motif in the 3′UTR region of indicated mRNA-encoding genes down-regulated in BCL9- knock out RKO cells. High fold change: Genes whose mRNA levels decreased more than 1.5-fold in BCL9 knockout cells. Low-fold change: others. P values were calculated using χ 2 test, ** P
Sfpq, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins"

Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins

Journal: Nature Communications

doi: 10.1038/s41467-019-13842-7

BCL9 regulates mRNA levels of calcium wave-associated genes through paraspeckle proteins. a Network of BCL9-interacting proteins identified by Co-IP MS. Each node represents a group of BCL9-interacting proteins with functional relationships. Lines between different nodes represent the “weight” of the protein-protein interaction according to String database (colored) or total peptides in Co-IP assay (black). Groups were clustered by k-means unsupervised classification according to the interacting “weight” among different nodes. b Representative IF showing co-localization of BCL9, NONO, and ILF2 in CRC but not in normal colon epithelial cells. Scale bars: 20 µm (top, left), and 2 µm (inset). c , d SFPQ binding motif in the 3′UTR region of indicated mRNA-encoding genes down-regulated in BCL9- knock out RKO cells. High fold change: Genes whose mRNA levels decreased more than 1.5-fold in BCL9 knockout cells. Low-fold change: others. P values were calculated using χ 2 test, ** P
Figure Legend Snippet: BCL9 regulates mRNA levels of calcium wave-associated genes through paraspeckle proteins. a Network of BCL9-interacting proteins identified by Co-IP MS. Each node represents a group of BCL9-interacting proteins with functional relationships. Lines between different nodes represent the “weight” of the protein-protein interaction according to String database (colored) or total peptides in Co-IP assay (black). Groups were clustered by k-means unsupervised classification according to the interacting “weight” among different nodes. b Representative IF showing co-localization of BCL9, NONO, and ILF2 in CRC but not in normal colon epithelial cells. Scale bars: 20 µm (top, left), and 2 µm (inset). c , d SFPQ binding motif in the 3′UTR region of indicated mRNA-encoding genes down-regulated in BCL9- knock out RKO cells. High fold change: Genes whose mRNA levels decreased more than 1.5-fold in BCL9 knockout cells. Low-fold change: others. P values were calculated using χ 2 test, ** P

Techniques Used: Co-Immunoprecipitation Assay, Mass Spectrometry, Functional Assay, Binding Assay, Knock-Out

2) Product Images from "BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins"

Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins

Journal: Nature Communications

doi: 10.1038/s41467-019-13842-7

BCL9 regulates mRNA levels of calcium wave-associated genes through paraspeckle proteins. a Network of BCL9-interacting proteins identified by Co-IP MS. Each node represents a group of BCL9-interacting proteins with functional relationships. Lines between different nodes represent the “weight” of the protein-protein interaction according to String database (colored) or total peptides in Co-IP assay (black). Groups were clustered by k-means unsupervised classification according to the interacting “weight” among different nodes. b Representative IF showing co-localization of BCL9, NONO, and ILF2 in CRC but not in normal colon epithelial cells. Scale bars: 20 µm (top, left), and 2 µm (inset). c , d SFPQ binding motif in the 3′UTR region of indicated mRNA-encoding genes down-regulated in BCL9- knock out RKO cells. High fold change: Genes whose mRNA levels decreased more than 1.5-fold in BCL9 knockout cells. Low-fold change: others. P values were calculated using χ 2 test, ** P
Figure Legend Snippet: BCL9 regulates mRNA levels of calcium wave-associated genes through paraspeckle proteins. a Network of BCL9-interacting proteins identified by Co-IP MS. Each node represents a group of BCL9-interacting proteins with functional relationships. Lines between different nodes represent the “weight” of the protein-protein interaction according to String database (colored) or total peptides in Co-IP assay (black). Groups were clustered by k-means unsupervised classification according to the interacting “weight” among different nodes. b Representative IF showing co-localization of BCL9, NONO, and ILF2 in CRC but not in normal colon epithelial cells. Scale bars: 20 µm (top, left), and 2 µm (inset). c , d SFPQ binding motif in the 3′UTR region of indicated mRNA-encoding genes down-regulated in BCL9- knock out RKO cells. High fold change: Genes whose mRNA levels decreased more than 1.5-fold in BCL9 knockout cells. Low-fold change: others. P values were calculated using χ 2 test, ** P

Techniques Used: Co-Immunoprecipitation Assay, Mass Spectrometry, Functional Assay, Binding Assay, Knock-Out

3) Product Images from "Nuclear matrix protein Matrin 3 is a regulator of ZAP-mediated retroviral restriction"

Article Title: Nuclear matrix protein Matrin 3 is a regulator of ZAP-mediated retroviral restriction

Journal: Retrovirology

doi: 10.1186/s12977-015-0182-4

Effects of other major nuclear matrix proteins on zap activity. Distinct nuclear matrix proteins regulate ZAP effects on a HIV-1 or HR’-CMV-luc and b MoMuLV. 293TrexhZAP2 cells were transfected with control siRNA (Ctrl) or siRNA against Matrin 3 (Matrin 3 KD), HNRNPU (HNRNPU KD), SFPQ (SFPQ KD), or Lamin A/C (Lamin A/C KD) for two consecutive days. Subsequently on the following day, cells were infected with HIV-luc or HR’-CMV-luc then treated with (ZAP) or without doxycycline treatment at 200 ng/ml. Cells were then harvested 2 days postinfection and lysates were subjected to luciferase assays to measure Nef-luc reporter activity. Fold restriction was calculated from dividing Ctrl siRNA values by the results of each treatment indicated. Data presented are the mean RLU/mg values of three independent experiments done in triplicate ±SD (HIV and MoMuLV) and two experiments done in triplicate ±SD for HR’-CMV-luc. Statistical analyses were done to test for differences between ZAP and ZAP induced in cells treated with siRNA. P values ≤0.05 are shown.
Figure Legend Snippet: Effects of other major nuclear matrix proteins on zap activity. Distinct nuclear matrix proteins regulate ZAP effects on a HIV-1 or HR’-CMV-luc and b MoMuLV. 293TrexhZAP2 cells were transfected with control siRNA (Ctrl) or siRNA against Matrin 3 (Matrin 3 KD), HNRNPU (HNRNPU KD), SFPQ (SFPQ KD), or Lamin A/C (Lamin A/C KD) for two consecutive days. Subsequently on the following day, cells were infected with HIV-luc or HR’-CMV-luc then treated with (ZAP) or without doxycycline treatment at 200 ng/ml. Cells were then harvested 2 days postinfection and lysates were subjected to luciferase assays to measure Nef-luc reporter activity. Fold restriction was calculated from dividing Ctrl siRNA values by the results of each treatment indicated. Data presented are the mean RLU/mg values of three independent experiments done in triplicate ±SD (HIV and MoMuLV) and two experiments done in triplicate ±SD for HR’-CMV-luc. Statistical analyses were done to test for differences between ZAP and ZAP induced in cells treated with siRNA. P values ≤0.05 are shown.

Techniques Used: Activity Assay, Transfection, Infection, Luciferase

Related Articles

Transfection:

Article Title: Nuclear matrix protein Matrin 3 is a regulator of ZAP-mediated retroviral restriction
Article Snippet: siRNA knockdown siRNA against Matrin 3 (Catalog #HSS114730, Invitrogen, Carlsbad, CA, USA), SFPQ (Catalog #HSS109642), HNRNPU (Catalog #HSS104917), Lamin A (Catalog #HSS106095), and negative control, Stealth RNAi siRNA Negative Control Med GC Duplex #2 (Invitrogen, Carlsbad, CA, USA) were used. .. 293TrexhZAP2 cells were transfected with siRNAs using Lipofectamine RNAiMAX (Invitrogen) according to manufacturer’s instructions.

Centrifugation:

Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins
Article Snippet: The membrane was subsequently probed using anti-BCL9 (1:1000 dilution, H00000607-M01, Abnova), NONO (1:1000 dilution, TA504777, Origene), SFPQ (1:1000 dilution, MA1-25325, ThermoFisher), ILF2 (1:5000 dilution, PA5-18718, ThermoFisher), or β-catenin (1:1000 dilution, 610154, BD Transduction Laboratories) antibodies. .. For total protein MS analysis, IP protein samples from anti-IgG and anti-BCL9 groups were recovered by Trichloroacetic acid (TCA 47658-U, Sigma) precipitation; samples were incubated for 10 min at 4°, before centrifugation at 16,000 × g for 5 min.

FLAG-tag:

Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins
Article Snippet: .. In all, 40 μg of each protein lysate was electrophoresed using SDS-PAGE, transferred to nitrocellulose membrane, blocked with 5% non-fat milk, and incubated overnight with following antibodies; anti-BCL9 (1:1000 dilution, ab37305, Abcam), NONO (1:1000 dilution, TA504777, Origene), SFPQ (1:1000 dilution, MA1-25325, ThermoFisher), ILF2 (1:5000 dilution, PA5-18718, ThermoFisher), TLR3 (1:500 dilution, ab62566, Abcam), β-catenin (1:1000 dilution, 610154, BD Transduction Laboratories), CD44(1:1000 dilution, #3570, Cell signaling), Flag tag (1:1000 dilution, A8592, sigma), Axin2 (1:1000 dilution, #2151, Cell signaling), p65 (1:1000 dilution, #8242, cell signaling), LaminB1 (1:1000 dilution, sc-6216, Santa Cruz), and GAPDH (1:1000 dilution, ab9485, Abcam). .. Secondary antibodies conjugated to horseradish peroxidase were purchased from Santa Cruz Biotechnology (1:5000 dilution, sc2020, Santa Cruz) and Cell signaling (1:5000 dilution, #7074, #7076, Cell signaling).

Incubation:

Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins
Article Snippet: .. In all, 40 μg of each protein lysate was electrophoresed using SDS-PAGE, transferred to nitrocellulose membrane, blocked with 5% non-fat milk, and incubated overnight with following antibodies; anti-BCL9 (1:1000 dilution, ab37305, Abcam), NONO (1:1000 dilution, TA504777, Origene), SFPQ (1:1000 dilution, MA1-25325, ThermoFisher), ILF2 (1:5000 dilution, PA5-18718, ThermoFisher), TLR3 (1:500 dilution, ab62566, Abcam), β-catenin (1:1000 dilution, 610154, BD Transduction Laboratories), CD44(1:1000 dilution, #3570, Cell signaling), Flag tag (1:1000 dilution, A8592, sigma), Axin2 (1:1000 dilution, #2151, Cell signaling), p65 (1:1000 dilution, #8242, cell signaling), LaminB1 (1:1000 dilution, sc-6216, Santa Cruz), and GAPDH (1:1000 dilution, ab9485, Abcam). .. Secondary antibodies conjugated to horseradish peroxidase were purchased from Santa Cruz Biotechnology (1:5000 dilution, sc2020, Santa Cruz) and Cell signaling (1:5000 dilution, #7074, #7076, Cell signaling).

Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins
Article Snippet: In all, 1 mg of the nuclear lysate was blocked with 5% BSA for 1 h at 4°, before overnight incubation with anti-BCL9 (1:500 dilution, ab37305 Abcam, 6109 generated in New England Biolabs), NONO (1:500 dilution, ab70335, Abcam), SFPQ (1:500 dilution, ab38148), ILF2 (1:500 dilution, H00003608-D01, Abnova), or β-catenin (1:500 dilution, 9562L, Cell signaling) antibodies. .. The membrane was subsequently probed using anti-BCL9 (1:1000 dilution, H00000607-M01, Abnova), NONO (1:1000 dilution, TA504777, Origene), SFPQ (1:1000 dilution, MA1-25325, ThermoFisher), ILF2 (1:5000 dilution, PA5-18718, ThermoFisher), or β-catenin (1:1000 dilution, 610154, BD Transduction Laboratories) antibodies.

Negative Control:

Article Title: Nuclear matrix protein Matrin 3 is a regulator of ZAP-mediated retroviral restriction
Article Snippet: .. siRNA knockdown siRNA against Matrin 3 (Catalog #HSS114730, Invitrogen, Carlsbad, CA, USA), SFPQ (Catalog #HSS109642), HNRNPU (Catalog #HSS104917), Lamin A (Catalog #HSS106095), and negative control, Stealth RNAi siRNA Negative Control Med GC Duplex #2 (Invitrogen, Carlsbad, CA, USA) were used. .. 293TrexhZAP2 cells were transfected with siRNAs using Lipofectamine RNAiMAX (Invitrogen) according to manufacturer’s instructions.

Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins
Article Snippet: Normal rabbit IgG (1:250 dilution, sc-3888, Santa Cruz) was used as a negative control. .. The membrane was subsequently probed using anti-BCL9 (1:1000 dilution, H00000607-M01, Abnova), NONO (1:1000 dilution, TA504777, Origene), SFPQ (1:1000 dilution, MA1-25325, ThermoFisher), ILF2 (1:5000 dilution, PA5-18718, ThermoFisher), or β-catenin (1:1000 dilution, 610154, BD Transduction Laboratories) antibodies.

Mass Spectrometry:

Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins
Article Snippet: The membrane was subsequently probed using anti-BCL9 (1:1000 dilution, H00000607-M01, Abnova), NONO (1:1000 dilution, TA504777, Origene), SFPQ (1:1000 dilution, MA1-25325, ThermoFisher), ILF2 (1:5000 dilution, PA5-18718, ThermoFisher), or β-catenin (1:1000 dilution, 610154, BD Transduction Laboratories) antibodies. .. For total protein MS analysis, IP protein samples from anti-IgG and anti-BCL9 groups were recovered by Trichloroacetic acid (TCA 47658-U, Sigma) precipitation; samples were incubated for 10 min at 4°, before centrifugation at 16,000 × g for 5 min.

Silver Staining:

Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins
Article Snippet: The membrane was subsequently probed using anti-BCL9 (1:1000 dilution, H00000607-M01, Abnova), NONO (1:1000 dilution, TA504777, Origene), SFPQ (1:1000 dilution, MA1-25325, ThermoFisher), ILF2 (1:5000 dilution, PA5-18718, ThermoFisher), or β-catenin (1:1000 dilution, 610154, BD Transduction Laboratories) antibodies. .. In addition, pulled down protein samples were also analyzed by silver staining; bands which existed in anti-BCL9 samples, but not in the anti-normal IgG groups, were cut and used for further MS analysis to validate our previous results.

SDS Page:

Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins
Article Snippet: .. In all, 40 μg of each protein lysate was electrophoresed using SDS-PAGE, transferred to nitrocellulose membrane, blocked with 5% non-fat milk, and incubated overnight with following antibodies; anti-BCL9 (1:1000 dilution, ab37305, Abcam), NONO (1:1000 dilution, TA504777, Origene), SFPQ (1:1000 dilution, MA1-25325, ThermoFisher), ILF2 (1:5000 dilution, PA5-18718, ThermoFisher), TLR3 (1:500 dilution, ab62566, Abcam), β-catenin (1:1000 dilution, 610154, BD Transduction Laboratories), CD44(1:1000 dilution, #3570, Cell signaling), Flag tag (1:1000 dilution, A8592, sigma), Axin2 (1:1000 dilution, #2151, Cell signaling), p65 (1:1000 dilution, #8242, cell signaling), LaminB1 (1:1000 dilution, sc-6216, Santa Cruz), and GAPDH (1:1000 dilution, ab9485, Abcam). .. Secondary antibodies conjugated to horseradish peroxidase were purchased from Santa Cruz Biotechnology (1:5000 dilution, sc2020, Santa Cruz) and Cell signaling (1:5000 dilution, #7074, #7076, Cell signaling).

Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins
Article Snippet: For Immunoblotting, the sample was electrophoresed using SDS-PAGE, transferred to nitrocellulose membrane and blocked using non-fat 5% milk. .. The membrane was subsequently probed using anti-BCL9 (1:1000 dilution, H00000607-M01, Abnova), NONO (1:1000 dilution, TA504777, Origene), SFPQ (1:1000 dilution, MA1-25325, ThermoFisher), ILF2 (1:5000 dilution, PA5-18718, ThermoFisher), or β-catenin (1:1000 dilution, 610154, BD Transduction Laboratories) antibodies.

Generated:

Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins
Article Snippet: In all, 1 mg of the nuclear lysate was blocked with 5% BSA for 1 h at 4°, before overnight incubation with anti-BCL9 (1:500 dilution, ab37305 Abcam, 6109 generated in New England Biolabs), NONO (1:500 dilution, ab70335, Abcam), SFPQ (1:500 dilution, ab38148), ILF2 (1:500 dilution, H00003608-D01, Abnova), or β-catenin (1:500 dilution, 9562L, Cell signaling) antibodies. .. The membrane was subsequently probed using anti-BCL9 (1:1000 dilution, H00000607-M01, Abnova), NONO (1:1000 dilution, TA504777, Origene), SFPQ (1:1000 dilution, MA1-25325, ThermoFisher), ILF2 (1:5000 dilution, PA5-18718, ThermoFisher), or β-catenin (1:1000 dilution, 610154, BD Transduction Laboratories) antibodies.

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    Thermo Fisher gene exp sfpq mm01179807 m1
    Derivation and validation of NONO-deficient MEFs. ( A ) Portion of mouse X chromosome depicting the Nono locus. Positions of gene trap within second intron and primers used for genotyping are shown. The mRNA product of the gene trap allele, which does not contain any of the Nono open reading frames, is shown in red. ( B ) Representative image showing genotyping by PCR. Left, analysis using primers P1 and P2 to detect wild-type allele; right, analysis using primers P3 and P4 to detect gene trap allele. In each panel, lane 1, wild-type male (+/0); lane 2, heterozygous female ( gt /+); lane 3, Nono-gene trap male ( gt /0). ( C ) Expression of NONO and β-geo gene trap mRNAs in tissues of mice sacrificed at postnatal day 4. Image shows PCR products with β-actin as a loading control. ( D ) NONO, <t>SFPQ</t> and internal reference protein (β-actin) expression in MEF isolates from mice bearing wild type (+) or gene trap ( gt ) alleles. Total cell lysates were probed with indicated antibodies. Left panel, gel image; right panel, quantification. ( E ) Immunostaining of the same four MEF isolates with anti-NONO antibody. Scale bar denotes 10 μm.
    Gene Exp Sfpq Mm01179807 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp sfpq mm01179807 m1/product/Thermo Fisher
    Average 89 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    89/100 stars
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    80
    Thermo Fisher sfpq
    <t>BCL9</t> regulates mRNA levels of calcium wave-associated genes through paraspeckle proteins. a Network of BCL9-interacting proteins identified by Co-IP MS. Each node represents a group of BCL9-interacting proteins with functional relationships. Lines between different nodes represent the “weight” of the protein-protein interaction according to String database (colored) or total peptides in Co-IP assay (black). Groups were clustered by k-means unsupervised classification according to the interacting “weight” among different nodes. b Representative IF showing co-localization of BCL9, NONO, and ILF2 in CRC but not in normal colon epithelial cells. Scale bars: 20 µm (top, left), and 2 µm (inset). c , d <t>SFPQ</t> binding motif in the 3′UTR region of indicated mRNA-encoding genes down-regulated in BCL9- knock out RKO cells. High fold change: Genes whose mRNA levels decreased more than 1.5-fold in BCL9 knockout cells. Low-fold change: others. P values were calculated using χ 2 test, ** P
    Sfpq, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sfpq/product/Thermo Fisher
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sfpq - by Bioz Stars, 2020-03
    80/100 stars
      Buy from Supplier

    85
    Thermo Fisher gene exp sfpq rn01179807 m1
    <t>SFPQ</t> binds motifs within the 3′UTRs of bclw and laminb2 ( a, b ) Schematic of rat laminb2 ( a ) and bclw ( b ) mRNA with SFPQ binding motifs 13 . Underlined motifs are conserved among rat, mouse and human. Western blot of proteins eluted from unbiotinylated mRNA ( un ), biotinylated gfp RNA ( gfp) , biotinylated 5′UTR ( 5 ′), open reading frame ( orf) or 3′UTR ( 3 ′) mRNA pull-down from sensory neuron protein lysates probed for SFPQ, FMRP and Musashi; data representative of 3 experiments. ( c ) Schematic of rat laminb2 3′UTR ( 3 ′ B ) with mutated SFPQ binding motifs 19 . Western blot of proteins eluted from unbiotinylated mRNA ( un ), biotinylated gfp mRNA ( gfp) , biotinylated 3′UTR ( 3 ′ B ) or biotinylated mutated 3′UTR ( 3 ′ B mut ) pull-down from sensory neuron protein lysates probed for SFPQ, FMRP and Musashi; data representative of 3 experiments. ( d ) Western blot following immunoprecipitation with α-SFPQ from sensory neuron protein lysate. Quantification of pull-down relative to input; data shows mean ± SEM, n = 3 experiments from cultures grown from 3 pregnant rats; * P = 0.0001, F (3,8) = 28.9 to noAb or KIF3B pull-down (one-way ANOVA with Bonferroni correction). Full-length blots are presented in Supplementary Figure 7 .
    Gene Exp Sfpq Rn01179807 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp sfpq rn01179807 m1/product/Thermo Fisher
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Derivation and validation of NONO-deficient MEFs. ( A ) Portion of mouse X chromosome depicting the Nono locus. Positions of gene trap within second intron and primers used for genotyping are shown. The mRNA product of the gene trap allele, which does not contain any of the Nono open reading frames, is shown in red. ( B ) Representative image showing genotyping by PCR. Left, analysis using primers P1 and P2 to detect wild-type allele; right, analysis using primers P3 and P4 to detect gene trap allele. In each panel, lane 1, wild-type male (+/0); lane 2, heterozygous female ( gt /+); lane 3, Nono-gene trap male ( gt /0). ( C ) Expression of NONO and β-geo gene trap mRNAs in tissues of mice sacrificed at postnatal day 4. Image shows PCR products with β-actin as a loading control. ( D ) NONO, SFPQ and internal reference protein (β-actin) expression in MEF isolates from mice bearing wild type (+) or gene trap ( gt ) alleles. Total cell lysates were probed with indicated antibodies. Left panel, gel image; right panel, quantification. ( E ) Immunostaining of the same four MEF isolates with anti-NONO antibody. Scale bar denotes 10 μm.

    Journal: Nucleic Acids Research

    Article Title: Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog

    doi: 10.1093/nar/gku650

    Figure Lengend Snippet: Derivation and validation of NONO-deficient MEFs. ( A ) Portion of mouse X chromosome depicting the Nono locus. Positions of gene trap within second intron and primers used for genotyping are shown. The mRNA product of the gene trap allele, which does not contain any of the Nono open reading frames, is shown in red. ( B ) Representative image showing genotyping by PCR. Left, analysis using primers P1 and P2 to detect wild-type allele; right, analysis using primers P3 and P4 to detect gene trap allele. In each panel, lane 1, wild-type male (+/0); lane 2, heterozygous female ( gt /+); lane 3, Nono-gene trap male ( gt /0). ( C ) Expression of NONO and β-geo gene trap mRNAs in tissues of mice sacrificed at postnatal day 4. Image shows PCR products with β-actin as a loading control. ( D ) NONO, SFPQ and internal reference protein (β-actin) expression in MEF isolates from mice bearing wild type (+) or gene trap ( gt ) alleles. Total cell lysates were probed with indicated antibodies. Left panel, gel image; right panel, quantification. ( E ) Immunostaining of the same four MEF isolates with anti-NONO antibody. Scale bar denotes 10 μm.

    Article Snippet: Taqman quantitative PCR assays were performed using the following probes (Life Technologies Corp.): Nono (Assay ID: Mm00834875_g1), Sfpq (Assay ID: Mm01179807_m1), Pspc1 (Assay ID: Mm00481804_m1), Prkdc (Assay ID: Mm01342967_m1), Xrcc5 (Assay ID: Mm00550142_m1), Xrcc6 (Assay ID: Mm00487458_m1), Lig4 (Assay ID: Mm01221720_m1), Nhej1 (Assay ID: Mm01259071_m1), Xrcc4 (Assay ID: Mm00459213_m1), Mre11a (Assay ID: Mm00450600_m1), Rad50 (Assay ID: Mm00485504_m1), Nbn (Assay ID: Mm00449854_m1), Rad51 (Assay ID: Mm00487905_m1), Rad51d (Assay ID: Rn01752219_m1) and internal control GAPDH (Assay ID: Mm99999915_g1).

    Techniques: Polymerase Chain Reaction, Expressing, Mouse Assay, Immunostaining

    Increased levels of PSPC1 and SFPQ–PSPC1 complex in NONO-deficient cells. ( A ) Immunostaining of WT-2 and gt -2 MEF isolates using anti-PSPC1 antibody. Scale bar, 10 μm. ( B ) Immunoblotting to determine levels of SFPQ, NONO and PSPC1 proteins in cells of indicated genotype. Two independent MEF populations, derived from different embryos, were analyzed for each type. Arrowheads denote proteins as indicated. ( C ) Quantification of data from panel (B). Values are normalized to β-actin. Error bars reflect standard deviation of values from independent MEF populations. ( D ) Immunoprecipitation (IP), followed by immunoblotting (IB), to protein–protein complexes in MEFs of indicated genotypes.

    Journal: Nucleic Acids Research

    Article Title: Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog

    doi: 10.1093/nar/gku650

    Figure Lengend Snippet: Increased levels of PSPC1 and SFPQ–PSPC1 complex in NONO-deficient cells. ( A ) Immunostaining of WT-2 and gt -2 MEF isolates using anti-PSPC1 antibody. Scale bar, 10 μm. ( B ) Immunoblotting to determine levels of SFPQ, NONO and PSPC1 proteins in cells of indicated genotype. Two independent MEF populations, derived from different embryos, were analyzed for each type. Arrowheads denote proteins as indicated. ( C ) Quantification of data from panel (B). Values are normalized to β-actin. Error bars reflect standard deviation of values from independent MEF populations. ( D ) Immunoprecipitation (IP), followed by immunoblotting (IB), to protein–protein complexes in MEFs of indicated genotypes.

    Article Snippet: Taqman quantitative PCR assays were performed using the following probes (Life Technologies Corp.): Nono (Assay ID: Mm00834875_g1), Sfpq (Assay ID: Mm01179807_m1), Pspc1 (Assay ID: Mm00481804_m1), Prkdc (Assay ID: Mm01342967_m1), Xrcc5 (Assay ID: Mm00550142_m1), Xrcc6 (Assay ID: Mm00487458_m1), Lig4 (Assay ID: Mm01221720_m1), Nhej1 (Assay ID: Mm01259071_m1), Xrcc4 (Assay ID: Mm00459213_m1), Mre11a (Assay ID: Mm00450600_m1), Rad50 (Assay ID: Mm00485504_m1), Nbn (Assay ID: Mm00449854_m1), Rad51 (Assay ID: Mm00487905_m1), Rad51d (Assay ID: Rn01752219_m1) and internal control GAPDH (Assay ID: Mm99999915_g1).

    Techniques: Immunostaining, Derivative Assay, Standard Deviation, Immunoprecipitation

    BCL9 regulates mRNA levels of calcium wave-associated genes through paraspeckle proteins. a Network of BCL9-interacting proteins identified by Co-IP MS. Each node represents a group of BCL9-interacting proteins with functional relationships. Lines between different nodes represent the “weight” of the protein-protein interaction according to String database (colored) or total peptides in Co-IP assay (black). Groups were clustered by k-means unsupervised classification according to the interacting “weight” among different nodes. b Representative IF showing co-localization of BCL9, NONO, and ILF2 in CRC but not in normal colon epithelial cells. Scale bars: 20 µm (top, left), and 2 µm (inset). c , d SFPQ binding motif in the 3′UTR region of indicated mRNA-encoding genes down-regulated in BCL9- knock out RKO cells. High fold change: Genes whose mRNA levels decreased more than 1.5-fold in BCL9 knockout cells. Low-fold change: others. P values were calculated using χ 2 test, ** P

    Journal: Nature Communications

    Article Title: BCL9 provides multi-cellular communication properties in colorectal cancer by interacting with paraspeckle proteins

    doi: 10.1038/s41467-019-13842-7

    Figure Lengend Snippet: BCL9 regulates mRNA levels of calcium wave-associated genes through paraspeckle proteins. a Network of BCL9-interacting proteins identified by Co-IP MS. Each node represents a group of BCL9-interacting proteins with functional relationships. Lines between different nodes represent the “weight” of the protein-protein interaction according to String database (colored) or total peptides in Co-IP assay (black). Groups were clustered by k-means unsupervised classification according to the interacting “weight” among different nodes. b Representative IF showing co-localization of BCL9, NONO, and ILF2 in CRC but not in normal colon epithelial cells. Scale bars: 20 µm (top, left), and 2 µm (inset). c , d SFPQ binding motif in the 3′UTR region of indicated mRNA-encoding genes down-regulated in BCL9- knock out RKO cells. High fold change: Genes whose mRNA levels decreased more than 1.5-fold in BCL9 knockout cells. Low-fold change: others. P values were calculated using χ 2 test, ** P

    Article Snippet: The membrane was subsequently probed using anti-BCL9 (1:1000 dilution, H00000607-M01, Abnova), NONO (1:1000 dilution, TA504777, Origene), SFPQ (1:1000 dilution, MA1-25325, ThermoFisher), ILF2 (1:5000 dilution, PA5-18718, ThermoFisher), or β-catenin (1:1000 dilution, 610154, BD Transduction Laboratories) antibodies.

    Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Functional Assay, Binding Assay, Knock-Out

    SFPQ binds motifs within the 3′UTRs of bclw and laminb2 ( a, b ) Schematic of rat laminb2 ( a ) and bclw ( b ) mRNA with SFPQ binding motifs 13 . Underlined motifs are conserved among rat, mouse and human. Western blot of proteins eluted from unbiotinylated mRNA ( un ), biotinylated gfp RNA ( gfp) , biotinylated 5′UTR ( 5 ′), open reading frame ( orf) or 3′UTR ( 3 ′) mRNA pull-down from sensory neuron protein lysates probed for SFPQ, FMRP and Musashi; data representative of 3 experiments. ( c ) Schematic of rat laminb2 3′UTR ( 3 ′ B ) with mutated SFPQ binding motifs 19 . Western blot of proteins eluted from unbiotinylated mRNA ( un ), biotinylated gfp mRNA ( gfp) , biotinylated 3′UTR ( 3 ′ B ) or biotinylated mutated 3′UTR ( 3 ′ B mut ) pull-down from sensory neuron protein lysates probed for SFPQ, FMRP and Musashi; data representative of 3 experiments. ( d ) Western blot following immunoprecipitation with α-SFPQ from sensory neuron protein lysate. Quantification of pull-down relative to input; data shows mean ± SEM, n = 3 experiments from cultures grown from 3 pregnant rats; * P = 0.0001, F (3,8) = 28.9 to noAb or KIF3B pull-down (one-way ANOVA with Bonferroni correction). Full-length blots are presented in Supplementary Figure 7 .

    Journal: Nature neuroscience

    Article Title: The RNA-binding protein SFPQ orchestrates an RNA regulon to promote axon viability

    doi: 10.1038/nn.4280

    Figure Lengend Snippet: SFPQ binds motifs within the 3′UTRs of bclw and laminb2 ( a, b ) Schematic of rat laminb2 ( a ) and bclw ( b ) mRNA with SFPQ binding motifs 13 . Underlined motifs are conserved among rat, mouse and human. Western blot of proteins eluted from unbiotinylated mRNA ( un ), biotinylated gfp RNA ( gfp) , biotinylated 5′UTR ( 5 ′), open reading frame ( orf) or 3′UTR ( 3 ′) mRNA pull-down from sensory neuron protein lysates probed for SFPQ, FMRP and Musashi; data representative of 3 experiments. ( c ) Schematic of rat laminb2 3′UTR ( 3 ′ B ) with mutated SFPQ binding motifs 19 . Western blot of proteins eluted from unbiotinylated mRNA ( un ), biotinylated gfp mRNA ( gfp) , biotinylated 3′UTR ( 3 ′ B ) or biotinylated mutated 3′UTR ( 3 ′ B mut ) pull-down from sensory neuron protein lysates probed for SFPQ, FMRP and Musashi; data representative of 3 experiments. ( d ) Western blot following immunoprecipitation with α-SFPQ from sensory neuron protein lysate. Quantification of pull-down relative to input; data shows mean ± SEM, n = 3 experiments from cultures grown from 3 pregnant rats; * P = 0.0001, F (3,8) = 28.9 to noAb or KIF3B pull-down (one-way ANOVA with Bonferroni correction). Full-length blots are presented in Supplementary Figure 7 .

    Article Snippet: Reverse transcription was performed using the cDNA archive kit (Applied Biosystems) according to the manufacturer’s specifications. qRT-PCR was performed using TaqMan gene expression assays (Applied Biosystems) to assess the RNA levels of rat bclw (Rn00821025_g1), laminB2 (Rn01408653_g1),β-actin (Rn00667869_m1), impa1 (Rn00583189_m1), creb (Rn00578829_g1), coxIV (Rn00665001_g1), smad5 (Rn00572484_m1), rpl4 (Rn00821091_g1) and sfpq (Rn01179807_m1).

    Techniques: Binding Assay, Western Blot, Immunoprecipitation

    SFPQ regulates localization of axonal mRNAs ( a ) qRT-PCR analysis of mRNA levels of distal axons from sensory neurons grown in compartmented cultures expressing SFPQ shRNA following 2h neurotrophin stimulation of distal axons (DA). Data shows mean ± SEM, n = 8 experiments from cultures grown from 8 pregnant rats; ** P

    Journal: Nature neuroscience

    Article Title: The RNA-binding protein SFPQ orchestrates an RNA regulon to promote axon viability

    doi: 10.1038/nn.4280

    Figure Lengend Snippet: SFPQ regulates localization of axonal mRNAs ( a ) qRT-PCR analysis of mRNA levels of distal axons from sensory neurons grown in compartmented cultures expressing SFPQ shRNA following 2h neurotrophin stimulation of distal axons (DA). Data shows mean ± SEM, n = 8 experiments from cultures grown from 8 pregnant rats; ** P

    Article Snippet: Reverse transcription was performed using the cDNA archive kit (Applied Biosystems) according to the manufacturer’s specifications. qRT-PCR was performed using TaqMan gene expression assays (Applied Biosystems) to assess the RNA levels of rat bclw (Rn00821025_g1), laminB2 (Rn01408653_g1),β-actin (Rn00667869_m1), impa1 (Rn00583189_m1), creb (Rn00578829_g1), coxIV (Rn00665001_g1), smad5 (Rn00572484_m1), rpl4 (Rn00821091_g1) and sfpq (Rn01179807_m1).

    Techniques: Quantitative RT-PCR, Expressing, shRNA

    SFPQ is required for axonal trafficking of laminb2 and bclw mRNA (a ) Subcellular fractionation of mRNA from nuclei (Nuc), cytoplasm (Cyt) and distal axons (DA) from sensory neurons grown in compartmented cultures. ( b–d ) qRT-PCR analysis of mRNA levels from Nuc, Cyt and DA from sensory neurons expressing SFPQ shRNA following 2h neurotrophin stimulation of DA. Data shows mean ± SEM, n = 8 experiments from cultures grown from 8 pregnant rats; * P

    Journal: Nature neuroscience

    Article Title: The RNA-binding protein SFPQ orchestrates an RNA regulon to promote axon viability

    doi: 10.1038/nn.4280

    Figure Lengend Snippet: SFPQ is required for axonal trafficking of laminb2 and bclw mRNA (a ) Subcellular fractionation of mRNA from nuclei (Nuc), cytoplasm (Cyt) and distal axons (DA) from sensory neurons grown in compartmented cultures. ( b–d ) qRT-PCR analysis of mRNA levels from Nuc, Cyt and DA from sensory neurons expressing SFPQ shRNA following 2h neurotrophin stimulation of DA. Data shows mean ± SEM, n = 8 experiments from cultures grown from 8 pregnant rats; * P

    Article Snippet: Reverse transcription was performed using the cDNA archive kit (Applied Biosystems) according to the manufacturer’s specifications. qRT-PCR was performed using TaqMan gene expression assays (Applied Biosystems) to assess the RNA levels of rat bclw (Rn00821025_g1), laminB2 (Rn01408653_g1),β-actin (Rn00667869_m1), impa1 (Rn00583189_m1), creb (Rn00578829_g1), coxIV (Rn00665001_g1), smad5 (Rn00572484_m1), rpl4 (Rn00821091_g1) and sfpq (Rn01179807_m1).

    Techniques: Fractionation, Quantitative RT-PCR, Expressing, shRNA

    SFPQ is required for co-assembly of bclw and laminb2 mRNA within RNA transport granules (a , b ) Representative single-molecule FISH for bclw and laminb2 mRNA ( a ) and bclw and γ-actin ( b ) of sensory neurons grown in microfluidic cultures, with DAPI (n = 3 individual microfluidic cultures). Scale bar, 5 μm. Zoom-in (white dotted area) shows colocalization of bclw and laminb2 mRNA (white arrow). Scale bar, 1 μm. ( c ) Super-resolution quantitative colocalization analysis of adjacent bclw and laminb2 mRNAs (blue; n = 131 cells) and adjacent bclw and γ-actin mRNAs (gray; n = 143 cells) within neuronal cell bodies in control conditions from 3 experiments. Normalized density is frequency of adjacent mRNAs at each distance on x-axis normalized to the frequency expected by chance. Shaded region shows 99% bootstrapped confidence intervals and red line is at y=1 (equal to chance) and x=270 nm. Colocalization of laminb2 and bclw mRNAs occurs 2x to 5x more often than would be expected by chance. ( d ) Super-resolution quantitative colocalization analysis (as shown in c ) in neuronal cell bodies expressing SFPQ shRNA; n = 112 cells from 3 experiments. ( e ) Percentage of cells exhibiting significant colocalization of bclw and laminb2 or bclw and γ-actin within 270 nm in control and shSFPQ conditions; n = 131 cells for Cntrl bclw and laminb2 , n = 143 cells for Cntrl bclw and γ-actin , n = 112 cells for shSFPQ bclw and laminb2 or γ-actin from 3 experiments, * P

    Journal: Nature neuroscience

    Article Title: The RNA-binding protein SFPQ orchestrates an RNA regulon to promote axon viability

    doi: 10.1038/nn.4280

    Figure Lengend Snippet: SFPQ is required for co-assembly of bclw and laminb2 mRNA within RNA transport granules (a , b ) Representative single-molecule FISH for bclw and laminb2 mRNA ( a ) and bclw and γ-actin ( b ) of sensory neurons grown in microfluidic cultures, with DAPI (n = 3 individual microfluidic cultures). Scale bar, 5 μm. Zoom-in (white dotted area) shows colocalization of bclw and laminb2 mRNA (white arrow). Scale bar, 1 μm. ( c ) Super-resolution quantitative colocalization analysis of adjacent bclw and laminb2 mRNAs (blue; n = 131 cells) and adjacent bclw and γ-actin mRNAs (gray; n = 143 cells) within neuronal cell bodies in control conditions from 3 experiments. Normalized density is frequency of adjacent mRNAs at each distance on x-axis normalized to the frequency expected by chance. Shaded region shows 99% bootstrapped confidence intervals and red line is at y=1 (equal to chance) and x=270 nm. Colocalization of laminb2 and bclw mRNAs occurs 2x to 5x more often than would be expected by chance. ( d ) Super-resolution quantitative colocalization analysis (as shown in c ) in neuronal cell bodies expressing SFPQ shRNA; n = 112 cells from 3 experiments. ( e ) Percentage of cells exhibiting significant colocalization of bclw and laminb2 or bclw and γ-actin within 270 nm in control and shSFPQ conditions; n = 131 cells for Cntrl bclw and laminb2 , n = 143 cells for Cntrl bclw and γ-actin , n = 112 cells for shSFPQ bclw and laminb2 or γ-actin from 3 experiments, * P

    Article Snippet: Reverse transcription was performed using the cDNA archive kit (Applied Biosystems) according to the manufacturer’s specifications. qRT-PCR was performed using TaqMan gene expression assays (Applied Biosystems) to assess the RNA levels of rat bclw (Rn00821025_g1), laminB2 (Rn01408653_g1),β-actin (Rn00667869_m1), impa1 (Rn00583189_m1), creb (Rn00578829_g1), coxIV (Rn00665001_g1), smad5 (Rn00572484_m1), rpl4 (Rn00821091_g1) and sfpq (Rn01179807_m1).

    Techniques: Fluorescence In Situ Hybridization, Expressing, shRNA

    SFPQ regulates functionally related genes to promote axonal viability ( a ) Western blot of protein from distal axon (DA) lysate from sensory neurons grown in compartmented cultures expressing control or SFPQ shRNA following 8h neurotrophin stimulation of DA. Quantification of LaminB2 and Bclw protein levels normalized to GAPDH; data shows mean ± SEM, n = 3; * P ≤ 0.05 (Paired two-tailed t -test; P = 0.05, t (2) = 2.84 for LaminB2 and P = 0.04, t (2) =3.29 for Bclw). Full-length blots are presented in Supplementary Figure 7 . ( b ) Representative immunostaining of sensory neuron axons with SFPQ, RPL17 and mitotracker (n = 3 individual neuronal cultures). Scale bar, 10 μm. ( c ) Representative binarized Tuj1-labeled axons in compartmented cultures expressing control (n = 56 axon tracts), SFPQ (n = 24 axon tracts) or LaminB2 (n = 32 axon tracts) shRNA in the absence (-NT) or presence (+NT) of neurotrophins (NGF+BDNF) from 3 individual experiments. Scale bar, 40 μm. ( d ) Quantification of axon degeneration; data shows mean values ± SEM, n = 25 axon tracts for shSFPQ+Bclw and n = 16 axon tracts for shLmnB2+Bclw from 3 experiments; * P

    Journal: Nature neuroscience

    Article Title: The RNA-binding protein SFPQ orchestrates an RNA regulon to promote axon viability

    doi: 10.1038/nn.4280

    Figure Lengend Snippet: SFPQ regulates functionally related genes to promote axonal viability ( a ) Western blot of protein from distal axon (DA) lysate from sensory neurons grown in compartmented cultures expressing control or SFPQ shRNA following 8h neurotrophin stimulation of DA. Quantification of LaminB2 and Bclw protein levels normalized to GAPDH; data shows mean ± SEM, n = 3; * P ≤ 0.05 (Paired two-tailed t -test; P = 0.05, t (2) = 2.84 for LaminB2 and P = 0.04, t (2) =3.29 for Bclw). Full-length blots are presented in Supplementary Figure 7 . ( b ) Representative immunostaining of sensory neuron axons with SFPQ, RPL17 and mitotracker (n = 3 individual neuronal cultures). Scale bar, 10 μm. ( c ) Representative binarized Tuj1-labeled axons in compartmented cultures expressing control (n = 56 axon tracts), SFPQ (n = 24 axon tracts) or LaminB2 (n = 32 axon tracts) shRNA in the absence (-NT) or presence (+NT) of neurotrophins (NGF+BDNF) from 3 individual experiments. Scale bar, 40 μm. ( d ) Quantification of axon degeneration; data shows mean values ± SEM, n = 25 axon tracts for shSFPQ+Bclw and n = 16 axon tracts for shLmnB2+Bclw from 3 experiments; * P

    Article Snippet: Reverse transcription was performed using the cDNA archive kit (Applied Biosystems) according to the manufacturer’s specifications. qRT-PCR was performed using TaqMan gene expression assays (Applied Biosystems) to assess the RNA levels of rat bclw (Rn00821025_g1), laminB2 (Rn01408653_g1),β-actin (Rn00667869_m1), impa1 (Rn00583189_m1), creb (Rn00578829_g1), coxIV (Rn00665001_g1), smad5 (Rn00572484_m1), rpl4 (Rn00821091_g1) and sfpq (Rn01179807_m1).

    Techniques: Western Blot, Expressing, shRNA, Two Tailed Test, Immunostaining, Labeling

    SFPQ binds axonal mRNAs ( a ) qRT-PCR analysis of SFPQ-precipitated mRNAs from Trk-PC12 cells following formaldehyde crosslinking. Number of SFPQ-binding motifs present within the 3′UTR of each mRNA is shown in the black box. Data, normalized to no antibody control, shows mean ± SEM, n = 4 crosslinking experiments; ** P

    Journal: Nature neuroscience

    Article Title: The RNA-binding protein SFPQ orchestrates an RNA regulon to promote axon viability

    doi: 10.1038/nn.4280

    Figure Lengend Snippet: SFPQ binds axonal mRNAs ( a ) qRT-PCR analysis of SFPQ-precipitated mRNAs from Trk-PC12 cells following formaldehyde crosslinking. Number of SFPQ-binding motifs present within the 3′UTR of each mRNA is shown in the black box. Data, normalized to no antibody control, shows mean ± SEM, n = 4 crosslinking experiments; ** P

    Article Snippet: Reverse transcription was performed using the cDNA archive kit (Applied Biosystems) according to the manufacturer’s specifications. qRT-PCR was performed using TaqMan gene expression assays (Applied Biosystems) to assess the RNA levels of rat bclw (Rn00821025_g1), laminB2 (Rn01408653_g1),β-actin (Rn00667869_m1), impa1 (Rn00583189_m1), creb (Rn00578829_g1), coxIV (Rn00665001_g1), smad5 (Rn00572484_m1), rpl4 (Rn00821091_g1) and sfpq (Rn01179807_m1).

    Techniques: Quantitative RT-PCR, Binding Assay