sox2 antibody  (Atlas Antibodies)

 
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    Name:
    Anti SOX2
    Description:
    SRY sex determining region Y box 2 Recombinant Protein Epitope Signature Tag PrEST antigen sequence
    Catalog Number:
    amab91307
    Price:
    None
    Applications:
    ICC-IF, IHC, WB
    Host:
    Mouse
    Conjugate:
    unconjugated
    Size:
    100 µl
    Category:
    Primary
    Isotype:
    IgG1
    Reactivity:
    Human Mouse
    Buy from Supplier


    Structured Review

    Atlas Antibodies sox2 antibody
    Anti SOX2
    SRY sex determining region Y box 2 Recombinant Protein Epitope Signature Tag PrEST antigen sequence
    https://www.bioz.com/result/sox2 antibody/product/Atlas Antibodies
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sox2 antibody - by Bioz Stars, 2020-07
    94/100 stars

    Images

    1) Product Images from "The Sox2 transcription factor binds RNA"

    Article Title: The Sox2 transcription factor binds RNA

    Journal: Nature Communications

    doi: 10.1038/s41467-020-15571-8

    Structure analysis of Sox2-HMG/nucleic acid-binding modes. a 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/DNA complex (red). The DNA sequence used in this experiment was derived from the FGF4 enhancer, corresponding to the sequence shown in Supplementary Table 1 . b 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/RNA complex (blue). The RNA sequence used in this experiment was the hairpin Loop B/Bulge(0 + 1) (Fig. 3c ).
    Figure Legend Snippet: Structure analysis of Sox2-HMG/nucleic acid-binding modes. a 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/DNA complex (red). The DNA sequence used in this experiment was derived from the FGF4 enhancer, corresponding to the sequence shown in Supplementary Table 1 . b 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/RNA complex (blue). The RNA sequence used in this experiment was the hairpin Loop B/Bulge(0 + 1) (Fig. 3c ).

    Techniques Used: Binding Assay, Nuclear Magnetic Resonance, Sequencing, Derivative Assay

    Characterization of the base-specific contributions to nucleic acid binding. a The relative binding affinities ( K D,rel ) for DNA and RNA are mapped to the Sox2-HMG surface (PDB ID 1GT0) according to the color scale to the left. b Plotted K D,rel values for DNA and RNA binding colored according to the scale in panel ( a ). The values of K D,rel are presented as the average and s.d. of n = 3 technical replicates. Bars above a gray background correspond to the Sox2-HMG major wing whereas a white background denotes the Sox2-HMG minor wing. P -values for the difference between K D,rel (DNA) and K D,rel (DNA) were calculated by the Student’s t -test assuming non-parametric distribution. All measured binding affinities and p -values for the alanine mutagenesis are provided in Supplementary Table 7 .
    Figure Legend Snippet: Characterization of the base-specific contributions to nucleic acid binding. a The relative binding affinities ( K D,rel ) for DNA and RNA are mapped to the Sox2-HMG surface (PDB ID 1GT0) according to the color scale to the left. b Plotted K D,rel values for DNA and RNA binding colored according to the scale in panel ( a ). The values of K D,rel are presented as the average and s.d. of n = 3 technical replicates. Bars above a gray background correspond to the Sox2-HMG major wing whereas a white background denotes the Sox2-HMG minor wing. P -values for the difference between K D,rel (DNA) and K D,rel (DNA) were calculated by the Student’s t -test assuming non-parametric distribution. All measured binding affinities and p -values for the alanine mutagenesis are provided in Supplementary Table 7 .

    Techniques Used: Binding Assay, RNA Binding Assay, Mutagenesis

    Sox2 interacts with RNA in mESC. a Schematic of fRIP and UV-RIP-Seq experimental flow. b , c Examples of enriched genes (Pmepa1 and Brd2) identified by both formaldehyde (“CH 2 O”) and UV-based RIP-seq. Each panel describes normalized coverage of sequencing reads (RPM: reads per million reads). d Correlation of enrichment between fRIP and UV-RIP. A dot represents a gene and each axis describes the fold change of IP relative to input in log scale. A color indicates statistical significance of enrichment (FDR-adjusted p -value
    Figure Legend Snippet: Sox2 interacts with RNA in mESC. a Schematic of fRIP and UV-RIP-Seq experimental flow. b , c Examples of enriched genes (Pmepa1 and Brd2) identified by both formaldehyde (“CH 2 O”) and UV-based RIP-seq. Each panel describes normalized coverage of sequencing reads (RPM: reads per million reads). d Correlation of enrichment between fRIP and UV-RIP. A dot represents a gene and each axis describes the fold change of IP relative to input in log scale. A color indicates statistical significance of enrichment (FDR-adjusted p -value

    Techniques Used: Sequencing

    Nucleic acid binding by Sox2 and Sox2-HMG. a Average binding curves of full-length Sox2 to nucleic acid ligands. Curves are presented as the average of normalized fraction bound with error bars displaying the standard deviation. b Figure legend and measured binding affinity values for panels ( a ) and ( c ). Values are presented as the average and s.e.m. of n = 4 technical replicates with the exception of Sox2-HMG/ ES2 which is the average and s.e.m. of n = 10 technical replicates. c Representative binding curves of the Sox2-HMG domain to nucleic acid ligands. Curves and error bars displayed as in panel ( a ).
    Figure Legend Snippet: Nucleic acid binding by Sox2 and Sox2-HMG. a Average binding curves of full-length Sox2 to nucleic acid ligands. Curves are presented as the average of normalized fraction bound with error bars displaying the standard deviation. b Figure legend and measured binding affinity values for panels ( a ) and ( c ). Values are presented as the average and s.e.m. of n = 4 technical replicates with the exception of Sox2-HMG/ ES2 which is the average and s.e.m. of n = 10 technical replicates. c Representative binding curves of the Sox2-HMG domain to nucleic acid ligands. Curves and error bars displayed as in panel ( a ).

    Techniques Used: Binding Assay, Standard Deviation

    Deletion analysis of the ES2 lncRNA. a Schematized depiction of the ES2 lncRNA and the segmented transcripts. Apparent binding affinity displayed as the average and s.e.m. of n = 6 technical replicates excluding 1–354 ES2, which is n = 10 technical replicates). b Sfold 44 predicted secondary structure of ES2 , nts 276–354, and minimized loop constructs. c Average binding curve for each Sox2-RNA interaction with error bars displaying the standard deviation of each measurements. K D,app is reported for the first transition and presented as the average and the s.e.m. of n = 6 technical replicates.
    Figure Legend Snippet: Deletion analysis of the ES2 lncRNA. a Schematized depiction of the ES2 lncRNA and the segmented transcripts. Apparent binding affinity displayed as the average and s.e.m. of n = 6 technical replicates excluding 1–354 ES2, which is n = 10 technical replicates). b Sfold 44 predicted secondary structure of ES2 , nts 276–354, and minimized loop constructs. c Average binding curve for each Sox2-RNA interaction with error bars displaying the standard deviation of each measurements. K D,app is reported for the first transition and presented as the average and the s.e.m. of n = 6 technical replicates.

    Techniques Used: Binding Assay, Construct, Standard Deviation

    Characterizing the nucleic acid-binding modes of Sox2-HMG. a Equilibrium competition binding assays measuring the anisotropy of the labeled nucleic acid as a function of unlabeled competitor. Binding curves presented as the average of n = 3 technical replicates with the error bars reflecting their s.d. b Determination of complex stoichiometry by EMSA. Migration of the radiolabeled nucleic acid species as a function of [Sox2-HMG]:[Ligand]. Bands are denoted as F (free), B1 (bound species 1), and B2 (bound species 2). Quantification provided in Supplementary Fig. 5 . c Analysis of the salt dependence of the Sox2-HMG/nucleic acid interactions. The colored bars show the measured binding affinities as the average of the n = 4 with the standard error reported (black shapes). The calculated slope of each linear regression is provided. d The calculated values of the electrostatic (Δ G el ) and non-electrostatic (Δ G nel ) components to binding the indicated nucleic acid ligand under the standard reaction conditions.
    Figure Legend Snippet: Characterizing the nucleic acid-binding modes of Sox2-HMG. a Equilibrium competition binding assays measuring the anisotropy of the labeled nucleic acid as a function of unlabeled competitor. Binding curves presented as the average of n = 3 technical replicates with the error bars reflecting their s.d. b Determination of complex stoichiometry by EMSA. Migration of the radiolabeled nucleic acid species as a function of [Sox2-HMG]:[Ligand]. Bands are denoted as F (free), B1 (bound species 1), and B2 (bound species 2). Quantification provided in Supplementary Fig. 5 . c Analysis of the salt dependence of the Sox2-HMG/nucleic acid interactions. The colored bars show the measured binding affinities as the average of the n = 4 with the standard error reported (black shapes). The calculated slope of each linear regression is provided. d The calculated values of the electrostatic (Δ G el ) and non-electrostatic (Δ G nel ) components to binding the indicated nucleic acid ligand under the standard reaction conditions.

    Techniques Used: Binding Assay, Labeling, Migration

    Interrogation of RNA features for high-affinity interaction with Sox2-HMG. a Secondary structure of the Loop B RNA with the designated paired regions (P1 and P2), internal loop (blue dotted line), and terminal loop (green dashed line). Binding assessed to mutations b reducing paired region helix length, n = 8, c reducing the internal loop size, n = 8, d truncation of hairpins, n = 3 e removing the terminal loop, n = 6, f structured RNAs, xpt riboswitch and env4 Cbl riboswitch, n = 4, tRNA Leu , n = 3, and g nucleic acid duplexes with n = 3 technical replicates each. Apparent binding affinity displayed as the average with s.e.m. reported.
    Figure Legend Snippet: Interrogation of RNA features for high-affinity interaction with Sox2-HMG. a Secondary structure of the Loop B RNA with the designated paired regions (P1 and P2), internal loop (blue dotted line), and terminal loop (green dashed line). Binding assessed to mutations b reducing paired region helix length, n = 8, c reducing the internal loop size, n = 8, d truncation of hairpins, n = 3 e removing the terminal loop, n = 6, f structured RNAs, xpt riboswitch and env4 Cbl riboswitch, n = 4, tRNA Leu , n = 3, and g nucleic acid duplexes with n = 3 technical replicates each. Apparent binding affinity displayed as the average with s.e.m. reported.

    Techniques Used: Binding Assay

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  • 94
    Atlas Antibodies sox2 antibody
    Structure analysis of <t>Sox2-HMG/nucleic</t> acid-binding modes. a 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/DNA complex (red). The DNA sequence used in this experiment was derived from the FGF4 enhancer, corresponding to the sequence shown in Supplementary Table 1 . b 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/RNA complex (blue). The RNA sequence used in this experiment was the hairpin Loop B/Bulge(0 + 1) (Fig. 3c ).
    Sox2 Antibody, supplied by Atlas Antibodies, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sox2 antibody/product/Atlas Antibodies
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sox2 antibody - by Bioz Stars, 2020-07
    94/100 stars
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    92
    Atlas Antibodies goat anti sox2
    Characterization of mouse in vitro differentiations in the mouse ES cell line carrying hChr21. (A) Expression of Sod1 and Son human genes located on Chromosome 21, the neural markers Tubb3 and Olig 1 and the mesoderm marker Meoxl in wt and hChr21 differentiations. (B) Percentage of OLIG2 positive cells from days 0 to 4 in wt and hChr21 differentiation show no major changes in cell proportions over time. (C) Representative images of smFISH used for segmentation and quantification of wt and hChr21 mouse cells. (D) Representative images of smFISH used for segmentation and quantification of human MN differentiation. (E) <t>Sox2</t> and total Olig2 mRNA number in human, wt and hChr21 neural progenitors. (F) hSOX2 and hOLIG2 mRNA number in human neural progenitors at days 4, 5 and 6. Scale bars 50μM.
    Goat Anti Sox2, supplied by Atlas Antibodies, used in various techniques. Bioz Stars score: 92/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti sox2/product/Atlas Antibodies
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti sox2 - by Bioz Stars, 2020-07
    92/100 stars
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    Structure analysis of Sox2-HMG/nucleic acid-binding modes. a 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/DNA complex (red). The DNA sequence used in this experiment was derived from the FGF4 enhancer, corresponding to the sequence shown in Supplementary Table 1 . b 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/RNA complex (blue). The RNA sequence used in this experiment was the hairpin Loop B/Bulge(0 + 1) (Fig. 3c ).

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Structure analysis of Sox2-HMG/nucleic acid-binding modes. a 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/DNA complex (red). The DNA sequence used in this experiment was derived from the FGF4 enhancer, corresponding to the sequence shown in Supplementary Table 1 . b 1 H– 15 N HSQC NMR spectrum of Sox2-HMG (black) overlaid with the spectrum of the Sox2-HMG/RNA complex (blue). The RNA sequence used in this experiment was the hairpin Loop B/Bulge(0 + 1) (Fig. 3c ).

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Binding Assay, Nuclear Magnetic Resonance, Sequencing, Derivative Assay

    Characterization of the base-specific contributions to nucleic acid binding. a The relative binding affinities ( K D,rel ) for DNA and RNA are mapped to the Sox2-HMG surface (PDB ID 1GT0) according to the color scale to the left. b Plotted K D,rel values for DNA and RNA binding colored according to the scale in panel ( a ). The values of K D,rel are presented as the average and s.d. of n = 3 technical replicates. Bars above a gray background correspond to the Sox2-HMG major wing whereas a white background denotes the Sox2-HMG minor wing. P -values for the difference between K D,rel (DNA) and K D,rel (DNA) were calculated by the Student’s t -test assuming non-parametric distribution. All measured binding affinities and p -values for the alanine mutagenesis are provided in Supplementary Table 7 .

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Characterization of the base-specific contributions to nucleic acid binding. a The relative binding affinities ( K D,rel ) for DNA and RNA are mapped to the Sox2-HMG surface (PDB ID 1GT0) according to the color scale to the left. b Plotted K D,rel values for DNA and RNA binding colored according to the scale in panel ( a ). The values of K D,rel are presented as the average and s.d. of n = 3 technical replicates. Bars above a gray background correspond to the Sox2-HMG major wing whereas a white background denotes the Sox2-HMG minor wing. P -values for the difference between K D,rel (DNA) and K D,rel (DNA) were calculated by the Student’s t -test assuming non-parametric distribution. All measured binding affinities and p -values for the alanine mutagenesis are provided in Supplementary Table 7 .

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Binding Assay, RNA Binding Assay, Mutagenesis

    Sox2 interacts with RNA in mESC. a Schematic of fRIP and UV-RIP-Seq experimental flow. b , c Examples of enriched genes (Pmepa1 and Brd2) identified by both formaldehyde (“CH 2 O”) and UV-based RIP-seq. Each panel describes normalized coverage of sequencing reads (RPM: reads per million reads). d Correlation of enrichment between fRIP and UV-RIP. A dot represents a gene and each axis describes the fold change of IP relative to input in log scale. A color indicates statistical significance of enrichment (FDR-adjusted p -value

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Sox2 interacts with RNA in mESC. a Schematic of fRIP and UV-RIP-Seq experimental flow. b , c Examples of enriched genes (Pmepa1 and Brd2) identified by both formaldehyde (“CH 2 O”) and UV-based RIP-seq. Each panel describes normalized coverage of sequencing reads (RPM: reads per million reads). d Correlation of enrichment between fRIP and UV-RIP. A dot represents a gene and each axis describes the fold change of IP relative to input in log scale. A color indicates statistical significance of enrichment (FDR-adjusted p -value

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Sequencing

    Nucleic acid binding by Sox2 and Sox2-HMG. a Average binding curves of full-length Sox2 to nucleic acid ligands. Curves are presented as the average of normalized fraction bound with error bars displaying the standard deviation. b Figure legend and measured binding affinity values for panels ( a ) and ( c ). Values are presented as the average and s.e.m. of n = 4 technical replicates with the exception of Sox2-HMG/ ES2 which is the average and s.e.m. of n = 10 technical replicates. c Representative binding curves of the Sox2-HMG domain to nucleic acid ligands. Curves and error bars displayed as in panel ( a ).

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Nucleic acid binding by Sox2 and Sox2-HMG. a Average binding curves of full-length Sox2 to nucleic acid ligands. Curves are presented as the average of normalized fraction bound with error bars displaying the standard deviation. b Figure legend and measured binding affinity values for panels ( a ) and ( c ). Values are presented as the average and s.e.m. of n = 4 technical replicates with the exception of Sox2-HMG/ ES2 which is the average and s.e.m. of n = 10 technical replicates. c Representative binding curves of the Sox2-HMG domain to nucleic acid ligands. Curves and error bars displayed as in panel ( a ).

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Binding Assay, Standard Deviation

    Deletion analysis of the ES2 lncRNA. a Schematized depiction of the ES2 lncRNA and the segmented transcripts. Apparent binding affinity displayed as the average and s.e.m. of n = 6 technical replicates excluding 1–354 ES2, which is n = 10 technical replicates). b Sfold 44 predicted secondary structure of ES2 , nts 276–354, and minimized loop constructs. c Average binding curve for each Sox2-RNA interaction with error bars displaying the standard deviation of each measurements. K D,app is reported for the first transition and presented as the average and the s.e.m. of n = 6 technical replicates.

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Deletion analysis of the ES2 lncRNA. a Schematized depiction of the ES2 lncRNA and the segmented transcripts. Apparent binding affinity displayed as the average and s.e.m. of n = 6 technical replicates excluding 1–354 ES2, which is n = 10 technical replicates). b Sfold 44 predicted secondary structure of ES2 , nts 276–354, and minimized loop constructs. c Average binding curve for each Sox2-RNA interaction with error bars displaying the standard deviation of each measurements. K D,app is reported for the first transition and presented as the average and the s.e.m. of n = 6 technical replicates.

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Binding Assay, Construct, Standard Deviation

    Characterizing the nucleic acid-binding modes of Sox2-HMG. a Equilibrium competition binding assays measuring the anisotropy of the labeled nucleic acid as a function of unlabeled competitor. Binding curves presented as the average of n = 3 technical replicates with the error bars reflecting their s.d. b Determination of complex stoichiometry by EMSA. Migration of the radiolabeled nucleic acid species as a function of [Sox2-HMG]:[Ligand]. Bands are denoted as F (free), B1 (bound species 1), and B2 (bound species 2). Quantification provided in Supplementary Fig. 5 . c Analysis of the salt dependence of the Sox2-HMG/nucleic acid interactions. The colored bars show the measured binding affinities as the average of the n = 4 with the standard error reported (black shapes). The calculated slope of each linear regression is provided. d The calculated values of the electrostatic (Δ G el ) and non-electrostatic (Δ G nel ) components to binding the indicated nucleic acid ligand under the standard reaction conditions.

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Characterizing the nucleic acid-binding modes of Sox2-HMG. a Equilibrium competition binding assays measuring the anisotropy of the labeled nucleic acid as a function of unlabeled competitor. Binding curves presented as the average of n = 3 technical replicates with the error bars reflecting their s.d. b Determination of complex stoichiometry by EMSA. Migration of the radiolabeled nucleic acid species as a function of [Sox2-HMG]:[Ligand]. Bands are denoted as F (free), B1 (bound species 1), and B2 (bound species 2). Quantification provided in Supplementary Fig. 5 . c Analysis of the salt dependence of the Sox2-HMG/nucleic acid interactions. The colored bars show the measured binding affinities as the average of the n = 4 with the standard error reported (black shapes). The calculated slope of each linear regression is provided. d The calculated values of the electrostatic (Δ G el ) and non-electrostatic (Δ G nel ) components to binding the indicated nucleic acid ligand under the standard reaction conditions.

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Binding Assay, Labeling, Migration

    Interrogation of RNA features for high-affinity interaction with Sox2-HMG. a Secondary structure of the Loop B RNA with the designated paired regions (P1 and P2), internal loop (blue dotted line), and terminal loop (green dashed line). Binding assessed to mutations b reducing paired region helix length, n = 8, c reducing the internal loop size, n = 8, d truncation of hairpins, n = 3 e removing the terminal loop, n = 6, f structured RNAs, xpt riboswitch and env4 Cbl riboswitch, n = 4, tRNA Leu , n = 3, and g nucleic acid duplexes with n = 3 technical replicates each. Apparent binding affinity displayed as the average with s.e.m. reported.

    Journal: Nature Communications

    Article Title: The Sox2 transcription factor binds RNA

    doi: 10.1038/s41467-020-15571-8

    Figure Lengend Snippet: Interrogation of RNA features for high-affinity interaction with Sox2-HMG. a Secondary structure of the Loop B RNA with the designated paired regions (P1 and P2), internal loop (blue dotted line), and terminal loop (green dashed line). Binding assessed to mutations b reducing paired region helix length, n = 8, c reducing the internal loop size, n = 8, d truncation of hairpins, n = 3 e removing the terminal loop, n = 6, f structured RNAs, xpt riboswitch and env4 Cbl riboswitch, n = 4, tRNA Leu , n = 3, and g nucleic acid duplexes with n = 3 technical replicates each. Apparent binding affinity displayed as the average with s.e.m. reported.

    Article Snippet: To the remaining supernatant, 5 µg of Sox2 antibody (Atlas Antibodies; validation shown in Supplementary Fig. ) was added and rotated for 2 h at 4 °C.

    Techniques: Binding Assay

    Characterization of mouse in vitro differentiations in the mouse ES cell line carrying hChr21. (A) Expression of Sod1 and Son human genes located on Chromosome 21, the neural markers Tubb3 and Olig 1 and the mesoderm marker Meoxl in wt and hChr21 differentiations. (B) Percentage of OLIG2 positive cells from days 0 to 4 in wt and hChr21 differentiation show no major changes in cell proportions over time. (C) Representative images of smFISH used for segmentation and quantification of wt and hChr21 mouse cells. (D) Representative images of smFISH used for segmentation and quantification of human MN differentiation. (E) Sox2 and total Olig2 mRNA number in human, wt and hChr21 neural progenitors. (F) hSOX2 and hOLIG2 mRNA number in human neural progenitors at days 4, 5 and 6. Scale bars 50μM.

    Journal: bioRxiv

    Article Title: Species-specific developmental timing is associated with global differences in protein stability in mouse and human

    doi: 10.1101/2019.12.29.889543

    Figure Lengend Snippet: Characterization of mouse in vitro differentiations in the mouse ES cell line carrying hChr21. (A) Expression of Sod1 and Son human genes located on Chromosome 21, the neural markers Tubb3 and Olig 1 and the mesoderm marker Meoxl in wt and hChr21 differentiations. (B) Percentage of OLIG2 positive cells from days 0 to 4 in wt and hChr21 differentiation show no major changes in cell proportions over time. (C) Representative images of smFISH used for segmentation and quantification of wt and hChr21 mouse cells. (D) Representative images of smFISH used for segmentation and quantification of human MN differentiation. (E) Sox2 and total Olig2 mRNA number in human, wt and hChr21 neural progenitors. (F) hSOX2 and hOLIG2 mRNA number in human neural progenitors at days 4, 5 and 6. Scale bars 50μM.

    Article Snippet: Primary antibodies were diluted as follows: rabbit anti-Pax6 (Covance 1:500), goat-anti Olig2 (R & D AF2418, 1:800), mouse anti-Nkx2.2 (,1:500), mouse anti-Hb9/Mnx1 (DSHB, 1:40), goat anti-Isl1 (R & D AF1837, 1:1,000), mouse anti-Hoxc6 (Santa Cruz Biotechnology sc-376330, 1:200), mouse anti-Tubb3 (Covance MMS-435P, 1:500), chicken anti-Tubb3 (Abcam ab107216, 1:500), goat anti-sox2 (R & D, 1:500), goat anti-sox9 (R & D AF3075, 1:250), rabbit anti-NFIA (Atlas antibodies HPA008884, 1:500).

    Techniques: In Vitro, Expressing, Marker

    Dynamics of Shh at the time of induction in mouse and human neural progenitors. (A) Percentage of NKX6.1 measured by flow cytometry in SOX2-e×pressing cells at day 2 and day 4 after addition of SAG at increasing concentrations. (B) NKX6.1 mean intensity level from the same samples with increasing concentrations of SAG are comparable at day 2 and day 4. (C) Scheme outlining the standard differentiation protocol where RA and SAG are added at the same time (light blue), versus a treatment where SAG addition is delayed for 24h (dark blue). (D) RT-qPCR data reveals no substantial differences in the induction dynamics of Gli1, Ptchi from the moment of addition of SAG. (E) RT-qPCR data measured in 12h intervals reveals similar gene expression dynamics in mouse (orange) and human (blue) Ptchi and distinct Irx3 expression. (F) Normalized expression of other Hedghog target genes from the RNAseq. (Transcripts per million (TPM)).

    Journal: bioRxiv

    Article Title: Species-specific developmental timing is associated with global differences in protein stability in mouse and human

    doi: 10.1101/2019.12.29.889543

    Figure Lengend Snippet: Dynamics of Shh at the time of induction in mouse and human neural progenitors. (A) Percentage of NKX6.1 measured by flow cytometry in SOX2-e×pressing cells at day 2 and day 4 after addition of SAG at increasing concentrations. (B) NKX6.1 mean intensity level from the same samples with increasing concentrations of SAG are comparable at day 2 and day 4. (C) Scheme outlining the standard differentiation protocol where RA and SAG are added at the same time (light blue), versus a treatment where SAG addition is delayed for 24h (dark blue). (D) RT-qPCR data reveals no substantial differences in the induction dynamics of Gli1, Ptchi from the moment of addition of SAG. (E) RT-qPCR data measured in 12h intervals reveals similar gene expression dynamics in mouse (orange) and human (blue) Ptchi and distinct Irx3 expression. (F) Normalized expression of other Hedghog target genes from the RNAseq. (Transcripts per million (TPM)).

    Article Snippet: Primary antibodies were diluted as follows: rabbit anti-Pax6 (Covance 1:500), goat-anti Olig2 (R & D AF2418, 1:800), mouse anti-Nkx2.2 (,1:500), mouse anti-Hb9/Mnx1 (DSHB, 1:40), goat anti-Isl1 (R & D AF1837, 1:1,000), mouse anti-Hoxc6 (Santa Cruz Biotechnology sc-376330, 1:200), mouse anti-Tubb3 (Covance MMS-435P, 1:500), chicken anti-Tubb3 (Abcam ab107216, 1:500), goat anti-sox2 (R & D, 1:500), goat anti-sox9 (R & D AF3075, 1:250), rabbit anti-NFIA (Atlas antibodies HPA008884, 1:500).

    Techniques: Flow Cytometry, Quantitative RT-PCR, Expressing

    Temporal control of gene expression depends on the species cellular environment. ( A ) Scatter plot with histograms of PAX6 and NKX6.1 expression measured by FACS in neural progenitors from wt (orange) and hChr21 containing (purple) mouse cells at day 2. ( B ) RT-qPCR analysis for the NP and neuronal markers Pax6, Nkx6.1, Nkx2.2 and Isl1 in wt and hChr21 lines. ( C ) RT-qPCR expression of Olig2 from the mouse (mOlig2) and human alleles (hOlig2) in wt and hChr21 lines. ( D ) smFISH at day 2 of differentiation in wt and hChr21 lines with probes for mSox2, and allele specific detection of mOlig2 or human Olig2 (hOLIG2). Scale bars = 10μm ( E ) Boxplots of RNA molecules per cell in wt and hChr21 cells using Sox2, Olig2 and human- and mouse-allele specific probes. ( F ) smFISH in human NPs at day 8 of differentiation for hSOX2 and hOLIG2. Scale bars = 50μm. ( G ) Boxplots of Sox2 and Olig2 mRNA molecules per cell in human NPs at day 8 and mouse wt and hChr21 cells at day 2.

    Journal: bioRxiv

    Article Title: Species-specific developmental timing is associated with global differences in protein stability in mouse and human

    doi: 10.1101/2019.12.29.889543

    Figure Lengend Snippet: Temporal control of gene expression depends on the species cellular environment. ( A ) Scatter plot with histograms of PAX6 and NKX6.1 expression measured by FACS in neural progenitors from wt (orange) and hChr21 containing (purple) mouse cells at day 2. ( B ) RT-qPCR analysis for the NP and neuronal markers Pax6, Nkx6.1, Nkx2.2 and Isl1 in wt and hChr21 lines. ( C ) RT-qPCR expression of Olig2 from the mouse (mOlig2) and human alleles (hOlig2) in wt and hChr21 lines. ( D ) smFISH at day 2 of differentiation in wt and hChr21 lines with probes for mSox2, and allele specific detection of mOlig2 or human Olig2 (hOLIG2). Scale bars = 10μm ( E ) Boxplots of RNA molecules per cell in wt and hChr21 cells using Sox2, Olig2 and human- and mouse-allele specific probes. ( F ) smFISH in human NPs at day 8 of differentiation for hSOX2 and hOLIG2. Scale bars = 50μm. ( G ) Boxplots of Sox2 and Olig2 mRNA molecules per cell in human NPs at day 8 and mouse wt and hChr21 cells at day 2.

    Article Snippet: Primary antibodies were diluted as follows: rabbit anti-Pax6 (Covance 1:500), goat-anti Olig2 (R & D AF2418, 1:800), mouse anti-Nkx2.2 (,1:500), mouse anti-Hb9/Mnx1 (DSHB, 1:40), goat anti-Isl1 (R & D AF1837, 1:1,000), mouse anti-Hoxc6 (Santa Cruz Biotechnology sc-376330, 1:200), mouse anti-Tubb3 (Covance MMS-435P, 1:500), chicken anti-Tubb3 (Abcam ab107216, 1:500), goat anti-sox2 (R & D, 1:500), goat anti-sox9 (R & D AF3075, 1:250), rabbit anti-NFIA (Atlas antibodies HPA008884, 1:500).

    Techniques: Expressing, FACS, Quantitative RT-PCR

    Comparison of neural tube development in mouse and human embryos ( A ) Scheme matching mouse and human neural tube development according to neural patterning and differentiation. ( B-D ) Immunofluorescence in transverse sections of mouse and human neural tubes at shoulder levels from E9.0 to E11.5 in mouse and CS11 to CS17 in human embryos. ( B ) Expression of progenitor markers PAX6 (green), OLIG2 (magenta) and NKX2.2 (cyan). ( C ) Pan-neural progenitor marker Sox2 (blue), and motor-neuron markers ISL1 (magenta) and HB9/MNX1 (cyan) at neurogenic stages. ( D ) Early ventral expression of gliogenic markers NFIA (red) and SOX9 (blue) in the neural tube can be detected from E10.5 in mouse and CS15 in human. NFIA also labels neurons, as indicated by TUBB3 (cyan) staining. Scale bars = 50 μm.

    Journal: bioRxiv

    Article Title: Species-specific developmental timing is associated with global differences in protein stability in mouse and human

    doi: 10.1101/2019.12.29.889543

    Figure Lengend Snippet: Comparison of neural tube development in mouse and human embryos ( A ) Scheme matching mouse and human neural tube development according to neural patterning and differentiation. ( B-D ) Immunofluorescence in transverse sections of mouse and human neural tubes at shoulder levels from E9.0 to E11.5 in mouse and CS11 to CS17 in human embryos. ( B ) Expression of progenitor markers PAX6 (green), OLIG2 (magenta) and NKX2.2 (cyan). ( C ) Pan-neural progenitor marker Sox2 (blue), and motor-neuron markers ISL1 (magenta) and HB9/MNX1 (cyan) at neurogenic stages. ( D ) Early ventral expression of gliogenic markers NFIA (red) and SOX9 (blue) in the neural tube can be detected from E10.5 in mouse and CS15 in human. NFIA also labels neurons, as indicated by TUBB3 (cyan) staining. Scale bars = 50 μm.

    Article Snippet: Primary antibodies were diluted as follows: rabbit anti-Pax6 (Covance 1:500), goat-anti Olig2 (R & D AF2418, 1:800), mouse anti-Nkx2.2 (,1:500), mouse anti-Hb9/Mnx1 (DSHB, 1:40), goat anti-Isl1 (R & D AF1837, 1:1,000), mouse anti-Hoxc6 (Santa Cruz Biotechnology sc-376330, 1:200), mouse anti-Tubb3 (Covance MMS-435P, 1:500), chicken anti-Tubb3 (Abcam ab107216, 1:500), goat anti-sox2 (R & D, 1:500), goat anti-sox9 (R & D AF3075, 1:250), rabbit anti-NFIA (Atlas antibodies HPA008884, 1:500).

    Techniques: Immunofluorescence, Expressing, Marker, Staining

    Characterization of a scaling factor for in vitro differentiations from mouse and human cells. (A) Expression of the neuromesodermal progenitor markers SOX2, CDX2, T at day 0 in mouse and human motor neuron differentiations. (B) Percentage of PAX6, NKX6.1 and OLIG2 positive cells at various time points across mouse and human differentiations. (C) Scatter plot with histograms of PAX6 and OLIG2 intensity levels measured by FACS in NPs from mouse cells at day 2 (orange) and human cells at days 4 (dark blue) and day 8 (light blue). (D) PCA plots of RNA-seq data across mouse and human MN differentiation. Shapes of the points indicate biological replicates. (E) Mean scaled expression profiles of selected human and mouse clusters pairs selected that share a higher proportion of homologous genes than expected by chance. (F) Estimated time factor on selected genes measured from the RNAseq dataset. (G) Expression of selected genes in time in mouse (solid orange line) and human differentiations (blue solid line) alognside with the rescaled expression in mouse (dashed orange line) using the estimated time factors for specific genes.

    Journal: bioRxiv

    Article Title: Species-specific developmental timing is associated with global differences in protein stability in mouse and human

    doi: 10.1101/2019.12.29.889543

    Figure Lengend Snippet: Characterization of a scaling factor for in vitro differentiations from mouse and human cells. (A) Expression of the neuromesodermal progenitor markers SOX2, CDX2, T at day 0 in mouse and human motor neuron differentiations. (B) Percentage of PAX6, NKX6.1 and OLIG2 positive cells at various time points across mouse and human differentiations. (C) Scatter plot with histograms of PAX6 and OLIG2 intensity levels measured by FACS in NPs from mouse cells at day 2 (orange) and human cells at days 4 (dark blue) and day 8 (light blue). (D) PCA plots of RNA-seq data across mouse and human MN differentiation. Shapes of the points indicate biological replicates. (E) Mean scaled expression profiles of selected human and mouse clusters pairs selected that share a higher proportion of homologous genes than expected by chance. (F) Estimated time factor on selected genes measured from the RNAseq dataset. (G) Expression of selected genes in time in mouse (solid orange line) and human differentiations (blue solid line) alognside with the rescaled expression in mouse (dashed orange line) using the estimated time factors for specific genes.

    Article Snippet: Primary antibodies were diluted as follows: rabbit anti-Pax6 (Covance 1:500), goat-anti Olig2 (R & D AF2418, 1:800), mouse anti-Nkx2.2 (,1:500), mouse anti-Hb9/Mnx1 (DSHB, 1:40), goat anti-Isl1 (R & D AF1837, 1:1,000), mouse anti-Hoxc6 (Santa Cruz Biotechnology sc-376330, 1:200), mouse anti-Tubb3 (Covance MMS-435P, 1:500), chicken anti-Tubb3 (Abcam ab107216, 1:500), goat anti-sox2 (R & D, 1:500), goat anti-sox9 (R & D AF3075, 1:250), rabbit anti-NFIA (Atlas antibodies HPA008884, 1:500).

    Techniques: In Vitro, Expressing, FACS, RNA Sequencing Assay

    Protein stability in the GRN accounts for the speed differences between species. (A) Schematic of EU experiment to measure RNA half-lives in mouse and human neural progenitors. Cells are cultured for 3h with the uriridine analog EU (green). A sample that contains maximal levels of EU incorpotation is collected (pulse). Subsequently, EU is removed and cells are collected at various time points. (B) Representative histograms of EU incorporation intensity measurements to estimate mRNA half-life from mouse day 2 (orange) and human day 4 (blue) neural progenitors. (C) Schematic of AHA experiment to measure protein half-lives in mouse and human neural progenitors. Cells are grown on methionine-free media for 45h and then cultured for 2h with the methionine analog AHA (green). A sample that contains maximal levels of AHA incorpotation is collected (pulse). Subsequently, AHA is removed and cells are collected at various time points. (D) Representative histograms of AHA incorporation intensity measurements to estimate protein half-life from mouse day 2 (orange) and human day 8 (blue) neural progenitors. (E) Normalized RT-qPCR analysis of RNA enrichment relative to Actin of EU-nascent RNA captured on magenetic beads after pulse and chase experiments. (F) Estimated time factor as a function of the degradation rate ratio accros various dorsoventral (DV) positions. (G) Representative mouse (G) and human (H) detection of NKX6.1, OLIG2, S0X1 and SOX2 after AHA pulse-chase experiments on nascent proteins.

    Journal: bioRxiv

    Article Title: Species-specific developmental timing is associated with global differences in protein stability in mouse and human

    doi: 10.1101/2019.12.29.889543

    Figure Lengend Snippet: Protein stability in the GRN accounts for the speed differences between species. (A) Schematic of EU experiment to measure RNA half-lives in mouse and human neural progenitors. Cells are cultured for 3h with the uriridine analog EU (green). A sample that contains maximal levels of EU incorpotation is collected (pulse). Subsequently, EU is removed and cells are collected at various time points. (B) Representative histograms of EU incorporation intensity measurements to estimate mRNA half-life from mouse day 2 (orange) and human day 4 (blue) neural progenitors. (C) Schematic of AHA experiment to measure protein half-lives in mouse and human neural progenitors. Cells are grown on methionine-free media for 45h and then cultured for 2h with the methionine analog AHA (green). A sample that contains maximal levels of AHA incorpotation is collected (pulse). Subsequently, AHA is removed and cells are collected at various time points. (D) Representative histograms of AHA incorporation intensity measurements to estimate protein half-life from mouse day 2 (orange) and human day 8 (blue) neural progenitors. (E) Normalized RT-qPCR analysis of RNA enrichment relative to Actin of EU-nascent RNA captured on magenetic beads after pulse and chase experiments. (F) Estimated time factor as a function of the degradation rate ratio accros various dorsoventral (DV) positions. (G) Representative mouse (G) and human (H) detection of NKX6.1, OLIG2, S0X1 and SOX2 after AHA pulse-chase experiments on nascent proteins.

    Article Snippet: Primary antibodies were diluted as follows: rabbit anti-Pax6 (Covance 1:500), goat-anti Olig2 (R & D AF2418, 1:800), mouse anti-Nkx2.2 (,1:500), mouse anti-Hb9/Mnx1 (DSHB, 1:40), goat anti-Isl1 (R & D AF1837, 1:1,000), mouse anti-Hoxc6 (Santa Cruz Biotechnology sc-376330, 1:200), mouse anti-Tubb3 (Covance MMS-435P, 1:500), chicken anti-Tubb3 (Abcam ab107216, 1:500), goat anti-sox2 (R & D, 1:500), goat anti-sox9 (R & D AF3075, 1:250), rabbit anti-NFIA (Atlas antibodies HPA008884, 1:500).

    Techniques: Cell Culture, Quantitative RT-PCR, Pulse Chase

    Protein degradation and cell cycle account for the speed differences between species. (A) Mouse and human normalized intensity measurements of NKX6.1, OLIG2, SOX1 and SOX2 after AHA pulse-chase experiments on AHA-labeled and purified nascent proteins. Line and shadowed areas show best exponential fit with 95% confidence intervals. (B) Normalized intensity measurements of mKATE2 in mouse and human Ptch1::T2A-mKate2 cell lines. Line and shadowed areas show best exponential fit and its 70% HDI. (C) Estimated half-lives for mKATE2 in mouse (orange) and human (blue) cells. (D) Cell cycle measurements of mouse neural progenitors at day two, and human neural progenitors at days 4 and 8. Line and shadowed areas show best fit and its 80% HDI (E) Cell cycle length estimations in mouse neural progenitors at day two, and human neural progenitors at days 4 and 8. For all plots, mouse data is orange-colored, and human is blue. Statistical significance (**) corresponds with

    Journal: bioRxiv

    Article Title: Species-specific developmental timing is associated with global differences in protein stability in mouse and human

    doi: 10.1101/2019.12.29.889543

    Figure Lengend Snippet: Protein degradation and cell cycle account for the speed differences between species. (A) Mouse and human normalized intensity measurements of NKX6.1, OLIG2, SOX1 and SOX2 after AHA pulse-chase experiments on AHA-labeled and purified nascent proteins. Line and shadowed areas show best exponential fit with 95% confidence intervals. (B) Normalized intensity measurements of mKATE2 in mouse and human Ptch1::T2A-mKate2 cell lines. Line and shadowed areas show best exponential fit and its 70% HDI. (C) Estimated half-lives for mKATE2 in mouse (orange) and human (blue) cells. (D) Cell cycle measurements of mouse neural progenitors at day two, and human neural progenitors at days 4 and 8. Line and shadowed areas show best fit and its 80% HDI (E) Cell cycle length estimations in mouse neural progenitors at day two, and human neural progenitors at days 4 and 8. For all plots, mouse data is orange-colored, and human is blue. Statistical significance (**) corresponds with

    Article Snippet: Primary antibodies were diluted as follows: rabbit anti-Pax6 (Covance 1:500), goat-anti Olig2 (R & D AF2418, 1:800), mouse anti-Nkx2.2 (,1:500), mouse anti-Hb9/Mnx1 (DSHB, 1:40), goat anti-Isl1 (R & D AF1837, 1:1,000), mouse anti-Hoxc6 (Santa Cruz Biotechnology sc-376330, 1:200), mouse anti-Tubb3 (Covance MMS-435P, 1:500), chicken anti-Tubb3 (Abcam ab107216, 1:500), goat anti-sox2 (R & D, 1:500), goat anti-sox9 (R & D AF3075, 1:250), rabbit anti-NFIA (Atlas antibodies HPA008884, 1:500).

    Techniques: Pulse Chase, Labeling, Purification