rabbit anti mi 2  (Santa Cruz Biotechnology)

 
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    Name:
    Mi2 Antibody
    Description:
    Anti Mi2 Antibody B 4 is a mouse monoclonal IgA kappa light chain Mi2 antibody provided at 200 µg ml raised against amino acids 1671 1912 of Mi2 of human origin recommended for detection of Mi2 α and Mi2 β of mouse rat and human origin by WB IP IF IHC P and ELISA also reactive with additional species including and equine canine and porcine Contact our Technical Service Department or your local Distributor for more information on how to receive a FREE 10 µg sample of Mi2 B 4 sc 55606
    Catalog Number:
    SC-55606
    Price:
    None
    Category:
    Antibodies Primary Antibodies and ImmunoCruz Conjugates Transcription Regulators Mi2 Antibodies Mi2 Antibody B 4
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    Structured Review

    Santa Cruz Biotechnology rabbit anti mi 2
    Effects of <t>Mi-2</t> depletion on histone modifications at the E(spl)-C locus. ( A ) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 or Hairless compared to control conditions (con: GFP RNAi). Mi-2 or Hairless knock down was performed for 3 days prior to the experiment. Note that Mi-2 and H are both downregulated indicating that the knockdown strategy was successful and E(spl)mδ, E(spl)mγ, E(spl)mα and E(spl)m7 are all significantly de-repressed in Notch-off state. ( B ) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 compared to control conditions (con: GFP RNAi) and to cells with Notch activation (Nact). Note that other Notch regulated genes such as CG12290 , CG17119, Notch and regular (rgl) are de-repressed in Notch-off state and have enhanced expression in Notch-on state ( C ) Fold change in RNA levels in Kc cells upon combined knockdown of Mi-2 and Hairless compared to control conditions and single knockdown of Mi-2 or Hairless . Note that Mi-2 and H are downregulated in both single and double conditions indicating that the knockdown strategy was successful. ( D ) Enrichment of Histone H3 is indicated at E(spl)-C or regions in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). H3 distribution in Notch active and in control conditions is not altered by knockdown of Mi-2 . ( E ) Enrichment for various histone modifications such as H3K27acetylation, H3K27trimethylation and H3K56acetylation is indicated at E(spl)-C locus in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). No change in the levels of these modifications was detected following knockdown of Mi-2 . (p values: *0.01
    Anti Mi2 Antibody B 4 is a mouse monoclonal IgA kappa light chain Mi2 antibody provided at 200 µg ml raised against amino acids 1671 1912 of Mi2 of human origin recommended for detection of Mi2 α and Mi2 β of mouse rat and human origin by WB IP IF IHC P and ELISA also reactive with additional species including and equine canine and porcine Contact our Technical Service Department or your local Distributor for more information on how to receive a FREE 10 µg sample of Mi2 B 4 sc 55606
    https://www.bioz.com/result/rabbit anti mi 2/product/Santa Cruz Biotechnology
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti mi 2 - by Bioz Stars, 2021-09
    92/100 stars

    Images

    1) Product Images from "Mi-2/NuRD complex protects stem cell progeny from mitogenic Notch signaling"

    Article Title: Mi-2/NuRD complex protects stem cell progeny from mitogenic Notch signaling

    Journal: eLife

    doi: 10.7554/eLife.41637

    Effects of Mi-2 depletion on histone modifications at the E(spl)-C locus. ( A ) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 or Hairless compared to control conditions (con: GFP RNAi). Mi-2 or Hairless knock down was performed for 3 days prior to the experiment. Note that Mi-2 and H are both downregulated indicating that the knockdown strategy was successful and E(spl)mδ, E(spl)mγ, E(spl)mα and E(spl)m7 are all significantly de-repressed in Notch-off state. ( B ) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 compared to control conditions (con: GFP RNAi) and to cells with Notch activation (Nact). Note that other Notch regulated genes such as CG12290 , CG17119, Notch and regular (rgl) are de-repressed in Notch-off state and have enhanced expression in Notch-on state ( C ) Fold change in RNA levels in Kc cells upon combined knockdown of Mi-2 and Hairless compared to control conditions and single knockdown of Mi-2 or Hairless . Note that Mi-2 and H are downregulated in both single and double conditions indicating that the knockdown strategy was successful. ( D ) Enrichment of Histone H3 is indicated at E(spl)-C or regions in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). H3 distribution in Notch active and in control conditions is not altered by knockdown of Mi-2 . ( E ) Enrichment for various histone modifications such as H3K27acetylation, H3K27trimethylation and H3K56acetylation is indicated at E(spl)-C locus in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). No change in the levels of these modifications was detected following knockdown of Mi-2 . (p values: *0.01
    Figure Legend Snippet: Effects of Mi-2 depletion on histone modifications at the E(spl)-C locus. ( A ) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 or Hairless compared to control conditions (con: GFP RNAi). Mi-2 or Hairless knock down was performed for 3 days prior to the experiment. Note that Mi-2 and H are both downregulated indicating that the knockdown strategy was successful and E(spl)mδ, E(spl)mγ, E(spl)mα and E(spl)m7 are all significantly de-repressed in Notch-off state. ( B ) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 compared to control conditions (con: GFP RNAi) and to cells with Notch activation (Nact). Note that other Notch regulated genes such as CG12290 , CG17119, Notch and regular (rgl) are de-repressed in Notch-off state and have enhanced expression in Notch-on state ( C ) Fold change in RNA levels in Kc cells upon combined knockdown of Mi-2 and Hairless compared to control conditions and single knockdown of Mi-2 or Hairless . Note that Mi-2 and H are downregulated in both single and double conditions indicating that the knockdown strategy was successful. ( D ) Enrichment of Histone H3 is indicated at E(spl)-C or regions in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). H3 distribution in Notch active and in control conditions is not altered by knockdown of Mi-2 . ( E ) Enrichment for various histone modifications such as H3K27acetylation, H3K27trimethylation and H3K56acetylation is indicated at E(spl)-C locus in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). No change in the levels of these modifications was detected following knockdown of Mi-2 . (p values: *0.01

    Techniques Used: Activation Assay, Expressing, Chromatin Immunoprecipitation

    Prospero does not cooperate with Mi-2 in NB lineages. ( A ) Depletion of Pros enhances the number of Dpn +ve cells (green or white channels) caused by Nact over-expression, Ase marks NBs and GMCs, Pros marks progeny. ( B ) Scatter dot plot, the number of Dpn +ve cells per VNC in > Nact; > Pros Ri (N = 24, three experiments) was significantly increased compared to > Nact; > w Ri larvae (N = 27, three experiments). ( C ) Overexpression of Pros ( > YFP Pros; w–Ri) results in decreased number of Dpn +ve cells per VNC (N = 15, four experiments) compared to wild type ( > LacZ; > w Ri; N = 17, four experiments) or Mi-2 knockdown alone ( > LacZ; > Mi-2-Ri; N = 16, four experiments). Concomitant loss of Mi-2 ( > YFP Pros; > Mi-2-Ri; N = 14, four experiments) does not rescue Pros-induced NB loss, instead it further decreases the number of Dpn +ve cells. Ase marks NBs and GMCs, Mira marks NBs. ( D ) Quantification of Dpn +ve cells in each condition, where shading of symbols indicates different experimental replicates for each genotype, with mean and SEM indicated by black lines.
    Figure Legend Snippet: Prospero does not cooperate with Mi-2 in NB lineages. ( A ) Depletion of Pros enhances the number of Dpn +ve cells (green or white channels) caused by Nact over-expression, Ase marks NBs and GMCs, Pros marks progeny. ( B ) Scatter dot plot, the number of Dpn +ve cells per VNC in > Nact; > Pros Ri (N = 24, three experiments) was significantly increased compared to > Nact; > w Ri larvae (N = 27, three experiments). ( C ) Overexpression of Pros ( > YFP Pros; w–Ri) results in decreased number of Dpn +ve cells per VNC (N = 15, four experiments) compared to wild type ( > LacZ; > w Ri; N = 17, four experiments) or Mi-2 knockdown alone ( > LacZ; > Mi-2-Ri; N = 16, four experiments). Concomitant loss of Mi-2 ( > YFP Pros; > Mi-2-Ri; N = 14, four experiments) does not rescue Pros-induced NB loss, instead it further decreases the number of Dpn +ve cells. Ase marks NBs and GMCs, Mira marks NBs. ( D ) Quantification of Dpn +ve cells in each condition, where shading of symbols indicates different experimental replicates for each genotype, with mean and SEM indicated by black lines.

    Techniques Used: Over Expression

    Model summarizing the role of Mi-2 in decommissioning Notch-responsive enhancers in NB progeny. The presence of Zfh1 and Mi-2 favours the decommissioning of enhancers from E(spl) and other Notch target genes in GMCs (yellow) to ensure their expression is switched off. Notch is on in NBs (green) and off in GMCs (yellow cells) due to asymmetrical segregation of Numb (ref). GMCs divide to produce two post-mitotic neuronal cells (grey). In NB lineages with constitutive Notch activity (Nact), the presence of Mi-2 at enhancers, recruited by Zfh1 (and potentially by other factors too), is sufficient to attenuate Nact, so that E(spl) and other target genes are switched off in GMCs. In a few NB progeny the effects of Mi-2 are overcome with time, and E(spl) genes are up-regulated as they revert to NB-like cells. Depletion of Mi-2 in NB lineages expressing Nact severely compromises enhancer decommissioning so that E(spl) and other Notch target genes are upregulated in many of the GMCs. The majority of the NB progeny acquire an NB-like fate. SUPPLEMENTARY MATERIAL: Legends for videos.
    Figure Legend Snippet: Model summarizing the role of Mi-2 in decommissioning Notch-responsive enhancers in NB progeny. The presence of Zfh1 and Mi-2 favours the decommissioning of enhancers from E(spl) and other Notch target genes in GMCs (yellow) to ensure their expression is switched off. Notch is on in NBs (green) and off in GMCs (yellow cells) due to asymmetrical segregation of Numb (ref). GMCs divide to produce two post-mitotic neuronal cells (grey). In NB lineages with constitutive Notch activity (Nact), the presence of Mi-2 at enhancers, recruited by Zfh1 (and potentially by other factors too), is sufficient to attenuate Nact, so that E(spl) and other target genes are switched off in GMCs. In a few NB progeny the effects of Mi-2 are overcome with time, and E(spl) genes are up-regulated as they revert to NB-like cells. Depletion of Mi-2 in NB lineages expressing Nact severely compromises enhancer decommissioning so that E(spl) and other Notch target genes are upregulated in many of the GMCs. The majority of the NB progeny acquire an NB-like fate. SUPPLEMENTARY MATERIAL: Legends for videos.

    Techniques Used: Expressing, Activity Assay

    Mi-2 depletion enhances the activation of Notch regulated genes. ( A–B ) Depletion of Mi-2 in NB lineages that receive excessive Notch signalling results in an increase in the expression of the Notch regulated enhancers from pathetic (A; path[NRE]-GFP, green) ( A ) or CycE (B; CycE[NRE]-GFP, green) as indicated. Expression of Dpn (red) and Notch reporters (green) in wild type (control), Nact expressing ( > Nact; > w-Ri) and Nact with Mi-2 depleted ( > Nact; > Mi-2-Ri). Hyperplastic lineages with low/no (arrows) or high (arrowheads) levels of Notch reporter expression are indicated. ( C ) Grainy head (Grh) and Miranda (Mira) are upregulated when Mi-2 is depleted in NB lineages expressing Nact; Dpn +ve (green), Grh +ve (red) and Mira +ve (blue) in the indicated genotypes.
    Figure Legend Snippet: Mi-2 depletion enhances the activation of Notch regulated genes. ( A–B ) Depletion of Mi-2 in NB lineages that receive excessive Notch signalling results in an increase in the expression of the Notch regulated enhancers from pathetic (A; path[NRE]-GFP, green) ( A ) or CycE (B; CycE[NRE]-GFP, green) as indicated. Expression of Dpn (red) and Notch reporters (green) in wild type (control), Nact expressing ( > Nact; > w-Ri) and Nact with Mi-2 depleted ( > Nact; > Mi-2-Ri). Hyperplastic lineages with low/no (arrows) or high (arrowheads) levels of Notch reporter expression are indicated. ( C ) Grainy head (Grh) and Miranda (Mira) are upregulated when Mi-2 is depleted in NB lineages expressing Nact; Dpn +ve (green), Grh +ve (red) and Mira +ve (blue) in the indicated genotypes.

    Techniques Used: Activation Assay, Expressing

    Mi-2 depletion in NBs, GMCs or newly born progeny exacerbates Notch-induced tumorigenesis but Mi-2 depletion in older neurons does not. ( A ) Mi-2 knockdown leads to severe depletion of Mi-2 protein levels. Mi-2 protein (red) levels in VNCs of the indicated genotypes: Nact alone ( grhNBGal4Gal80ts > NΔecd; > w- Ri), Mi-2 knockdown in Nact ( > NΔecd; > Mi-2-Ri ) or Mi-2 knockdown alone ( > LacZ; > Mi-2-Ri ). Dpn (green) and Mira (blue) mark NBs. ( B ) Depletion of Mi-2 in NBs using insc-Gal4 leads to an increase of the number of hyperplastic Dpn +ve cells in Nact conditions. Expression of Dpn (green or white) is shown in VNCs with Nact alone ( > NΔecd; > w- Ri) and with knock down of Mi-2 in combination with Nact ( > NΔecd; > Mi-2-Ri ). Ase (red) marks NBs and GMCs, whereas Mira (blue) marks NBs ( C ) Depletion of Mi-2 in GMCs and newly born NSC progeny using cas GMR71C09 -Gal4 leads to an increase in the number of hyperplastic Dpn +ve cells in Nact conditions. Dpn (white) in wild type ( > LacZ; > w- Ri), Nact expressing ( > NΔecd; > w- Ri) knock down of Mi-2 only ( > LacZ; > Mi-2-Ri Bloomington line #33415), and knock down of Mi-2 in Nact expressing ( > NΔecd; > Mi-2-Ri ) lineages. Ase (red) marks NBs and GMCs, whereas Mira (blue) marks NBs. ( D ) Neither Nact nor Mi-2 depletion nor combination of Nact with Mi-2 depletion in mature neurons via nsyb-Gal4 lead to increase in Dpn +ve cells. Dpn (white) in knock-down of Mi-2 only ( > LacZ; > Mi-2-Ri ; Bloomington line #33415), Nact expressing ( > NΔecd; > w- Ri) and knock down of Mi-2 in Nact expressing ( > NΔecd; > Mi-2-Ri ) lineages. Ase (red) marks NBs and GMCs, whereas Mira (blue) marks NBs. ( E ). Quantification of the number of Dpn +ve cells per VNC under the conditions indicated. When Mi-2 was depleted in GMCs using cas GMR71C09 -Gal4 in Nact conditions compared to Nact alone the number of Dpn +ve cells per VNC was significantly increased. VNCs from WT or Mi-2 knockdown backgrounds exhibit normal number of Dpn +ve cells. Box represents IQR, black line indicates median and whiskers indicate ±1.5 × IQR. (*p
    Figure Legend Snippet: Mi-2 depletion in NBs, GMCs or newly born progeny exacerbates Notch-induced tumorigenesis but Mi-2 depletion in older neurons does not. ( A ) Mi-2 knockdown leads to severe depletion of Mi-2 protein levels. Mi-2 protein (red) levels in VNCs of the indicated genotypes: Nact alone ( grhNBGal4Gal80ts > NΔecd; > w- Ri), Mi-2 knockdown in Nact ( > NΔecd; > Mi-2-Ri ) or Mi-2 knockdown alone ( > LacZ; > Mi-2-Ri ). Dpn (green) and Mira (blue) mark NBs. ( B ) Depletion of Mi-2 in NBs using insc-Gal4 leads to an increase of the number of hyperplastic Dpn +ve cells in Nact conditions. Expression of Dpn (green or white) is shown in VNCs with Nact alone ( > NΔecd; > w- Ri) and with knock down of Mi-2 in combination with Nact ( > NΔecd; > Mi-2-Ri ). Ase (red) marks NBs and GMCs, whereas Mira (blue) marks NBs ( C ) Depletion of Mi-2 in GMCs and newly born NSC progeny using cas GMR71C09 -Gal4 leads to an increase in the number of hyperplastic Dpn +ve cells in Nact conditions. Dpn (white) in wild type ( > LacZ; > w- Ri), Nact expressing ( > NΔecd; > w- Ri) knock down of Mi-2 only ( > LacZ; > Mi-2-Ri Bloomington line #33415), and knock down of Mi-2 in Nact expressing ( > NΔecd; > Mi-2-Ri ) lineages. Ase (red) marks NBs and GMCs, whereas Mira (blue) marks NBs. ( D ) Neither Nact nor Mi-2 depletion nor combination of Nact with Mi-2 depletion in mature neurons via nsyb-Gal4 lead to increase in Dpn +ve cells. Dpn (white) in knock-down of Mi-2 only ( > LacZ; > Mi-2-Ri ; Bloomington line #33415), Nact expressing ( > NΔecd; > w- Ri) and knock down of Mi-2 in Nact expressing ( > NΔecd; > Mi-2-Ri ) lineages. Ase (red) marks NBs and GMCs, whereas Mira (blue) marks NBs. ( E ). Quantification of the number of Dpn +ve cells per VNC under the conditions indicated. When Mi-2 was depleted in GMCs using cas GMR71C09 -Gal4 in Nact conditions compared to Nact alone the number of Dpn +ve cells per VNC was significantly increased. VNCs from WT or Mi-2 knockdown backgrounds exhibit normal number of Dpn +ve cells. Box represents IQR, black line indicates median and whiskers indicate ±1.5 × IQR. (*p

    Techniques Used: Expressing

    Expression of Ε(spl)mγ-GFP in GMCs upon Mi-2 depletion in NB lineages. ( A ) Time points from a time lapse movie of LacZ; Mi-2-Ri NB lineages (dissociated from larval brains with grhNBGal4 Gal80ts at the permissive temperature for 24 hr). Ε(spl)mγ-GFP is switched off in emerging GMCs as in WT lineages. Upper panels, bright field images of NB and its progeny combined with Histone-RFP (red, H2Av-RFP) to monitor cell cycle stages; lower panels, expression of Ε(spl)mγ-GFP (black). Time is depicted below each panel along with cartoons of the lineages where NBs are in darker shades of green, emerging GMCs with low expression of Ε(spl)mγ-GFP in light green and GMCs with no expression of E(spl)mγ-GFP in yellow. Asterisks indicate progeny from same ancestor cell. Numbers correspond to different cells indexed in B. Scale bar 25 μm. ( B ) Bar chart depicting progression of each cell in a Mi-2-Ri NB lineage, bar thickness indicates cell-size and the colour represents Ε(spl)mγ-GFP levels according to the scale (blue low levels, yellow high levels). Dashed lines mark mitotic events linking to the emerging daughter cells. ( C ) Plot of Ε(spl)mγ-GFP levels in NBs, newly born GMCs and progeny in > LacZ; Mi-2-Ri . Note that Ε(spl)mγ-GFP levels decay in newly born GMCS in > LacZ; Mi-2-Ri lineages.
    Figure Legend Snippet: Expression of Ε(spl)mγ-GFP in GMCs upon Mi-2 depletion in NB lineages. ( A ) Time points from a time lapse movie of LacZ; Mi-2-Ri NB lineages (dissociated from larval brains with grhNBGal4 Gal80ts at the permissive temperature for 24 hr). Ε(spl)mγ-GFP is switched off in emerging GMCs as in WT lineages. Upper panels, bright field images of NB and its progeny combined with Histone-RFP (red, H2Av-RFP) to monitor cell cycle stages; lower panels, expression of Ε(spl)mγ-GFP (black). Time is depicted below each panel along with cartoons of the lineages where NBs are in darker shades of green, emerging GMCs with low expression of Ε(spl)mγ-GFP in light green and GMCs with no expression of E(spl)mγ-GFP in yellow. Asterisks indicate progeny from same ancestor cell. Numbers correspond to different cells indexed in B. Scale bar 25 μm. ( B ) Bar chart depicting progression of each cell in a Mi-2-Ri NB lineage, bar thickness indicates cell-size and the colour represents Ε(spl)mγ-GFP levels according to the scale (blue low levels, yellow high levels). Dashed lines mark mitotic events linking to the emerging daughter cells. ( C ) Plot of Ε(spl)mγ-GFP levels in NBs, newly born GMCs and progeny in > LacZ; Mi-2-Ri . Note that Ε(spl)mγ-GFP levels decay in newly born GMCS in > LacZ; Mi-2-Ri lineages.

    Techniques Used: Expressing

    Zfh1 resists Notch-induced t umorigenesis. ( A ) Ectopic expression of Zfh1 reduces the number of Dpn +ve cells (green or white channels) caused by Nact overexpression. This effect is partially rescued by the loss of Mi-2. Ase marks NBs and GMCs, Pros marks progeny. ( B ) Quantification of Dpn +ve cells per VNC in each condition, where shading of symbols indicates different experimental replicates for each genotype, mean and SEM indicated by black lines. > Nact; > zfh1 PB, > w Ri (N = 26, three experiments) was significantly decreased compared to > Nact; > LacZ, > w Ri larvae (N = 11, three experiments), whereas it remained unaltered in > Nact; > zfh1 PB, > Mi-2-Ri (N = 20, three experiments). ( C ) Mi-2-YFP and zfh1-GFP expression in type II NB lineages. Mi-2 is expressed in high levels in the Type II NB and lower levels in INPs, whereas Zfh1 is expressed only in some neurons. Yellow outlines of Type II lineages. Dpn (red) and Mira (blue) mark neuroblasts and INPs.
    Figure Legend Snippet: Zfh1 resists Notch-induced t umorigenesis. ( A ) Ectopic expression of Zfh1 reduces the number of Dpn +ve cells (green or white channels) caused by Nact overexpression. This effect is partially rescued by the loss of Mi-2. Ase marks NBs and GMCs, Pros marks progeny. ( B ) Quantification of Dpn +ve cells per VNC in each condition, where shading of symbols indicates different experimental replicates for each genotype, mean and SEM indicated by black lines. > Nact; > zfh1 PB, > w Ri (N = 26, three experiments) was significantly decreased compared to > Nact; > LacZ, > w Ri larvae (N = 11, three experiments), whereas it remained unaltered in > Nact; > zfh1 PB, > Mi-2-Ri (N = 20, three experiments). ( C ) Mi-2-YFP and zfh1-GFP expression in type II NB lineages. Mi-2 is expressed in high levels in the Type II NB and lower levels in INPs, whereas Zfh1 is expressed only in some neurons. Yellow outlines of Type II lineages. Dpn (red) and Mira (blue) mark neuroblasts and INPs.

    Techniques Used: Expressing, Over Expression

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    Incubation:

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    Binding Assay:

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    Fluorescence In Situ Hybridization:

    Article Title: CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality
    Article Snippet: .. Reagents and antibodies Gateway® BP Clonase® Enzyme Mix and Gateway® LR Clonase® Enzyme mix (Life Technologies); anti HDAC1 ab (Santa Cruz: SC-7872); anti MTA2 ab (Abcam: ab8106); antiRbAp46 ab (Abcam: ab3535); anti Tubulin ab [DM1A] (Abcam: ab7291); anti CHD3 ab and anti CHD4N ab (Prof. Weidong Wang, National Institute on aging, Baltimore, Maryland, USA or ( )); anti Mi-2 antibody H-242 (Santa-Cruz: sc-11378 X); anti HP-1 α ab (Merck Millipore: MAB3446); anti HP-1 β ab (Abcam: ab10478); anti HP-1 γ ab (Merck Millipore: MAB3450); HRP anti rabbit (Jackson Immuno Research Laboratories Inc.: 111-035-144) and HRP anti mouse (Jackson Immuno Research Laboratories Inc.: 115-035-146); mouse monoclonal anti rabbit IgG light chain HRP conjugated ab (Abcam ab 99697) and mouse monoclonal anti rabbit IgG heavy chain HRP conjugated ab (Abcam ab 99072); Alexa 594-anti rabbit (Molecular Probes: A-11012); GFP-Trap® _A Beads and Binding control-agarose beads for preclearing (from Chromotek); ANTI-FLAG® M2 Affinity Gel and FLAG® Peptide (Sigma Aldrich); HiTrap SP FF and HiLoad 16/60 Superdex 200 (Amersham); Protein A-Sepharose GE CL-4B 17-0780-01 (Sigma Aldrich); Amicon Ultra Centrifugal Filter Unit [MWCO 10 kDa] (Millipore); (protease free, fatty acid free, essentially globulin free) BSA (Sigma Aldrich); gelatin from cold water fish skin (Sigma Aldrich); NuPAGE® Novex® 4–12% Bis-Tris Protein Gels, 1.0 mm, 10 well (Life Technologies); NucleoSpin RNA Kit (Macherey-Nagel); TruSeq RNA Sample Preparation Kit v2 (Illumina); iScript™ cDNA Synthesis Kit (Bio-Rad); FCS [‘Tetracycline free’] (Biochrom); FCS (Gibco); DMEM (1x) + GlutaMAX™-I [1g/l glucose] (Life Technologies); Gibco Sf-900™ II SFM (1×) Serum free Medium (Life Technologies); Doxycycline (Clontech Laboratories, Inc.); Zeozin (Life Technologies), Hygromycin (Life Technologies), Blasticidin (Life Technologies); FuGENE® HD transfection reagent (Promega); Effectene transfection reagent (Qiagen); HotStarTaq® Plus DNA Polymerase (Qiagen); SuperSignal West Dura Extended Duration Substrate and SuperSignal West Femto Trial Kit (Thermo Scientific); Triton X-100 (Sigma Aldrich); Igepal CA-630 (Sigma Aldrich); Tween-20 (Roth); Hydroxylapatite, Fast Flow (Calbiochem); 2-Mercaptoethanol (Merck); PEG4000 (Roth); Trypsin Gold, mass spectrometry grade (Promega); 32 P-γ-ATP (Hartmann Analytic); Benzonase ≥ 250 U/μl (E1014 Sigma-Aldrich); Ethidium bromide (Roth). ..

    Sample Prep:

    Article Title: CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality
    Article Snippet: .. Reagents and antibodies Gateway® BP Clonase® Enzyme Mix and Gateway® LR Clonase® Enzyme mix (Life Technologies); anti HDAC1 ab (Santa Cruz: SC-7872); anti MTA2 ab (Abcam: ab8106); antiRbAp46 ab (Abcam: ab3535); anti Tubulin ab [DM1A] (Abcam: ab7291); anti CHD3 ab and anti CHD4N ab (Prof. Weidong Wang, National Institute on aging, Baltimore, Maryland, USA or ( )); anti Mi-2 antibody H-242 (Santa-Cruz: sc-11378 X); anti HP-1 α ab (Merck Millipore: MAB3446); anti HP-1 β ab (Abcam: ab10478); anti HP-1 γ ab (Merck Millipore: MAB3450); HRP anti rabbit (Jackson Immuno Research Laboratories Inc.: 111-035-144) and HRP anti mouse (Jackson Immuno Research Laboratories Inc.: 115-035-146); mouse monoclonal anti rabbit IgG light chain HRP conjugated ab (Abcam ab 99697) and mouse monoclonal anti rabbit IgG heavy chain HRP conjugated ab (Abcam ab 99072); Alexa 594-anti rabbit (Molecular Probes: A-11012); GFP-Trap® _A Beads and Binding control-agarose beads for preclearing (from Chromotek); ANTI-FLAG® M2 Affinity Gel and FLAG® Peptide (Sigma Aldrich); HiTrap SP FF and HiLoad 16/60 Superdex 200 (Amersham); Protein A-Sepharose GE CL-4B 17-0780-01 (Sigma Aldrich); Amicon Ultra Centrifugal Filter Unit [MWCO 10 kDa] (Millipore); (protease free, fatty acid free, essentially globulin free) BSA (Sigma Aldrich); gelatin from cold water fish skin (Sigma Aldrich); NuPAGE® Novex® 4–12% Bis-Tris Protein Gels, 1.0 mm, 10 well (Life Technologies); NucleoSpin RNA Kit (Macherey-Nagel); TruSeq RNA Sample Preparation Kit v2 (Illumina); iScript™ cDNA Synthesis Kit (Bio-Rad); FCS [‘Tetracycline free’] (Biochrom); FCS (Gibco); DMEM (1x) + GlutaMAX™-I [1g/l glucose] (Life Technologies); Gibco Sf-900™ II SFM (1×) Serum free Medium (Life Technologies); Doxycycline (Clontech Laboratories, Inc.); Zeozin (Life Technologies), Hygromycin (Life Technologies), Blasticidin (Life Technologies); FuGENE® HD transfection reagent (Promega); Effectene transfection reagent (Qiagen); HotStarTaq® Plus DNA Polymerase (Qiagen); SuperSignal West Dura Extended Duration Substrate and SuperSignal West Femto Trial Kit (Thermo Scientific); Triton X-100 (Sigma Aldrich); Igepal CA-630 (Sigma Aldrich); Tween-20 (Roth); Hydroxylapatite, Fast Flow (Calbiochem); 2-Mercaptoethanol (Merck); PEG4000 (Roth); Trypsin Gold, mass spectrometry grade (Promega); 32 P-γ-ATP (Hartmann Analytic); Benzonase ≥ 250 U/μl (E1014 Sigma-Aldrich); Ethidium bromide (Roth). ..

    Transfection:

    Article Title: CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality
    Article Snippet: .. Reagents and antibodies Gateway® BP Clonase® Enzyme Mix and Gateway® LR Clonase® Enzyme mix (Life Technologies); anti HDAC1 ab (Santa Cruz: SC-7872); anti MTA2 ab (Abcam: ab8106); antiRbAp46 ab (Abcam: ab3535); anti Tubulin ab [DM1A] (Abcam: ab7291); anti CHD3 ab and anti CHD4N ab (Prof. Weidong Wang, National Institute on aging, Baltimore, Maryland, USA or ( )); anti Mi-2 antibody H-242 (Santa-Cruz: sc-11378 X); anti HP-1 α ab (Merck Millipore: MAB3446); anti HP-1 β ab (Abcam: ab10478); anti HP-1 γ ab (Merck Millipore: MAB3450); HRP anti rabbit (Jackson Immuno Research Laboratories Inc.: 111-035-144) and HRP anti mouse (Jackson Immuno Research Laboratories Inc.: 115-035-146); mouse monoclonal anti rabbit IgG light chain HRP conjugated ab (Abcam ab 99697) and mouse monoclonal anti rabbit IgG heavy chain HRP conjugated ab (Abcam ab 99072); Alexa 594-anti rabbit (Molecular Probes: A-11012); GFP-Trap® _A Beads and Binding control-agarose beads for preclearing (from Chromotek); ANTI-FLAG® M2 Affinity Gel and FLAG® Peptide (Sigma Aldrich); HiTrap SP FF and HiLoad 16/60 Superdex 200 (Amersham); Protein A-Sepharose GE CL-4B 17-0780-01 (Sigma Aldrich); Amicon Ultra Centrifugal Filter Unit [MWCO 10 kDa] (Millipore); (protease free, fatty acid free, essentially globulin free) BSA (Sigma Aldrich); gelatin from cold water fish skin (Sigma Aldrich); NuPAGE® Novex® 4–12% Bis-Tris Protein Gels, 1.0 mm, 10 well (Life Technologies); NucleoSpin RNA Kit (Macherey-Nagel); TruSeq RNA Sample Preparation Kit v2 (Illumina); iScript™ cDNA Synthesis Kit (Bio-Rad); FCS [‘Tetracycline free’] (Biochrom); FCS (Gibco); DMEM (1x) + GlutaMAX™-I [1g/l glucose] (Life Technologies); Gibco Sf-900™ II SFM (1×) Serum free Medium (Life Technologies); Doxycycline (Clontech Laboratories, Inc.); Zeozin (Life Technologies), Hygromycin (Life Technologies), Blasticidin (Life Technologies); FuGENE® HD transfection reagent (Promega); Effectene transfection reagent (Qiagen); HotStarTaq® Plus DNA Polymerase (Qiagen); SuperSignal West Dura Extended Duration Substrate and SuperSignal West Femto Trial Kit (Thermo Scientific); Triton X-100 (Sigma Aldrich); Igepal CA-630 (Sigma Aldrich); Tween-20 (Roth); Hydroxylapatite, Fast Flow (Calbiochem); 2-Mercaptoethanol (Merck); PEG4000 (Roth); Trypsin Gold, mass spectrometry grade (Promega); 32 P-γ-ATP (Hartmann Analytic); Benzonase ≥ 250 U/μl (E1014 Sigma-Aldrich); Ethidium bromide (Roth). ..

    Flow Cytometry:

    Article Title: CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality
    Article Snippet: .. Reagents and antibodies Gateway® BP Clonase® Enzyme Mix and Gateway® LR Clonase® Enzyme mix (Life Technologies); anti HDAC1 ab (Santa Cruz: SC-7872); anti MTA2 ab (Abcam: ab8106); antiRbAp46 ab (Abcam: ab3535); anti Tubulin ab [DM1A] (Abcam: ab7291); anti CHD3 ab and anti CHD4N ab (Prof. Weidong Wang, National Institute on aging, Baltimore, Maryland, USA or ( )); anti Mi-2 antibody H-242 (Santa-Cruz: sc-11378 X); anti HP-1 α ab (Merck Millipore: MAB3446); anti HP-1 β ab (Abcam: ab10478); anti HP-1 γ ab (Merck Millipore: MAB3450); HRP anti rabbit (Jackson Immuno Research Laboratories Inc.: 111-035-144) and HRP anti mouse (Jackson Immuno Research Laboratories Inc.: 115-035-146); mouse monoclonal anti rabbit IgG light chain HRP conjugated ab (Abcam ab 99697) and mouse monoclonal anti rabbit IgG heavy chain HRP conjugated ab (Abcam ab 99072); Alexa 594-anti rabbit (Molecular Probes: A-11012); GFP-Trap® _A Beads and Binding control-agarose beads for preclearing (from Chromotek); ANTI-FLAG® M2 Affinity Gel and FLAG® Peptide (Sigma Aldrich); HiTrap SP FF and HiLoad 16/60 Superdex 200 (Amersham); Protein A-Sepharose GE CL-4B 17-0780-01 (Sigma Aldrich); Amicon Ultra Centrifugal Filter Unit [MWCO 10 kDa] (Millipore); (protease free, fatty acid free, essentially globulin free) BSA (Sigma Aldrich); gelatin from cold water fish skin (Sigma Aldrich); NuPAGE® Novex® 4–12% Bis-Tris Protein Gels, 1.0 mm, 10 well (Life Technologies); NucleoSpin RNA Kit (Macherey-Nagel); TruSeq RNA Sample Preparation Kit v2 (Illumina); iScript™ cDNA Synthesis Kit (Bio-Rad); FCS [‘Tetracycline free’] (Biochrom); FCS (Gibco); DMEM (1x) + GlutaMAX™-I [1g/l glucose] (Life Technologies); Gibco Sf-900™ II SFM (1×) Serum free Medium (Life Technologies); Doxycycline (Clontech Laboratories, Inc.); Zeozin (Life Technologies), Hygromycin (Life Technologies), Blasticidin (Life Technologies); FuGENE® HD transfection reagent (Promega); Effectene transfection reagent (Qiagen); HotStarTaq® Plus DNA Polymerase (Qiagen); SuperSignal West Dura Extended Duration Substrate and SuperSignal West Femto Trial Kit (Thermo Scientific); Triton X-100 (Sigma Aldrich); Igepal CA-630 (Sigma Aldrich); Tween-20 (Roth); Hydroxylapatite, Fast Flow (Calbiochem); 2-Mercaptoethanol (Merck); PEG4000 (Roth); Trypsin Gold, mass spectrometry grade (Promega); 32 P-γ-ATP (Hartmann Analytic); Benzonase ≥ 250 U/μl (E1014 Sigma-Aldrich); Ethidium bromide (Roth). ..

    Mass Spectrometry:

    Article Title: CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality
    Article Snippet: .. Reagents and antibodies Gateway® BP Clonase® Enzyme Mix and Gateway® LR Clonase® Enzyme mix (Life Technologies); anti HDAC1 ab (Santa Cruz: SC-7872); anti MTA2 ab (Abcam: ab8106); antiRbAp46 ab (Abcam: ab3535); anti Tubulin ab [DM1A] (Abcam: ab7291); anti CHD3 ab and anti CHD4N ab (Prof. Weidong Wang, National Institute on aging, Baltimore, Maryland, USA or ( )); anti Mi-2 antibody H-242 (Santa-Cruz: sc-11378 X); anti HP-1 α ab (Merck Millipore: MAB3446); anti HP-1 β ab (Abcam: ab10478); anti HP-1 γ ab (Merck Millipore: MAB3450); HRP anti rabbit (Jackson Immuno Research Laboratories Inc.: 111-035-144) and HRP anti mouse (Jackson Immuno Research Laboratories Inc.: 115-035-146); mouse monoclonal anti rabbit IgG light chain HRP conjugated ab (Abcam ab 99697) and mouse monoclonal anti rabbit IgG heavy chain HRP conjugated ab (Abcam ab 99072); Alexa 594-anti rabbit (Molecular Probes: A-11012); GFP-Trap® _A Beads and Binding control-agarose beads for preclearing (from Chromotek); ANTI-FLAG® M2 Affinity Gel and FLAG® Peptide (Sigma Aldrich); HiTrap SP FF and HiLoad 16/60 Superdex 200 (Amersham); Protein A-Sepharose GE CL-4B 17-0780-01 (Sigma Aldrich); Amicon Ultra Centrifugal Filter Unit [MWCO 10 kDa] (Millipore); (protease free, fatty acid free, essentially globulin free) BSA (Sigma Aldrich); gelatin from cold water fish skin (Sigma Aldrich); NuPAGE® Novex® 4–12% Bis-Tris Protein Gels, 1.0 mm, 10 well (Life Technologies); NucleoSpin RNA Kit (Macherey-Nagel); TruSeq RNA Sample Preparation Kit v2 (Illumina); iScript™ cDNA Synthesis Kit (Bio-Rad); FCS [‘Tetracycline free’] (Biochrom); FCS (Gibco); DMEM (1x) + GlutaMAX™-I [1g/l glucose] (Life Technologies); Gibco Sf-900™ II SFM (1×) Serum free Medium (Life Technologies); Doxycycline (Clontech Laboratories, Inc.); Zeozin (Life Technologies), Hygromycin (Life Technologies), Blasticidin (Life Technologies); FuGENE® HD transfection reagent (Promega); Effectene transfection reagent (Qiagen); HotStarTaq® Plus DNA Polymerase (Qiagen); SuperSignal West Dura Extended Duration Substrate and SuperSignal West Femto Trial Kit (Thermo Scientific); Triton X-100 (Sigma Aldrich); Igepal CA-630 (Sigma Aldrich); Tween-20 (Roth); Hydroxylapatite, Fast Flow (Calbiochem); 2-Mercaptoethanol (Merck); PEG4000 (Roth); Trypsin Gold, mass spectrometry grade (Promega); 32 P-γ-ATP (Hartmann Analytic); Benzonase ≥ 250 U/μl (E1014 Sigma-Aldrich); Ethidium bromide (Roth). ..

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    Santa Cruz Biotechnology rabbit anti mi 2
    Effects of <t>Mi-2</t> depletion on histone modifications at the E(spl)-C locus. ( A ) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 or Hairless compared to control conditions (con: GFP RNAi). Mi-2 or Hairless knock down was performed for 3 days prior to the experiment. Note that Mi-2 and H are both downregulated indicating that the knockdown strategy was successful and E(spl)mδ, E(spl)mγ, E(spl)mα and E(spl)m7 are all significantly de-repressed in Notch-off state. ( B ) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 compared to control conditions (con: GFP RNAi) and to cells with Notch activation (Nact). Note that other Notch regulated genes such as CG12290 , CG17119, Notch and regular (rgl) are de-repressed in Notch-off state and have enhanced expression in Notch-on state ( C ) Fold change in RNA levels in Kc cells upon combined knockdown of Mi-2 and Hairless compared to control conditions and single knockdown of Mi-2 or Hairless . Note that Mi-2 and H are downregulated in both single and double conditions indicating that the knockdown strategy was successful. ( D ) Enrichment of Histone H3 is indicated at E(spl)-C or regions in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). H3 distribution in Notch active and in control conditions is not altered by knockdown of Mi-2 . ( E ) Enrichment for various histone modifications such as H3K27acetylation, H3K27trimethylation and H3K56acetylation is indicated at E(spl)-C locus in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). No change in the levels of these modifications was detected following knockdown of Mi-2 . (p values: *0.01
    Rabbit Anti Mi 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of Mi-2 depletion on histone modifications at the E(spl)-C locus. ( A ) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 or Hairless compared to control conditions (con: GFP RNAi). Mi-2 or Hairless knock down was performed for 3 days prior to the experiment. Note that Mi-2 and H are both downregulated indicating that the knockdown strategy was successful and E(spl)mδ, E(spl)mγ, E(spl)mα and E(spl)m7 are all significantly de-repressed in Notch-off state. ( B ) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 compared to control conditions (con: GFP RNAi) and to cells with Notch activation (Nact). Note that other Notch regulated genes such as CG12290 , CG17119, Notch and regular (rgl) are de-repressed in Notch-off state and have enhanced expression in Notch-on state ( C ) Fold change in RNA levels in Kc cells upon combined knockdown of Mi-2 and Hairless compared to control conditions and single knockdown of Mi-2 or Hairless . Note that Mi-2 and H are downregulated in both single and double conditions indicating that the knockdown strategy was successful. ( D ) Enrichment of Histone H3 is indicated at E(spl)-C or regions in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). H3 distribution in Notch active and in control conditions is not altered by knockdown of Mi-2 . ( E ) Enrichment for various histone modifications such as H3K27acetylation, H3K27trimethylation and H3K56acetylation is indicated at E(spl)-C locus in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). No change in the levels of these modifications was detected following knockdown of Mi-2 . (p values: *0.01

    Journal: eLife

    Article Title: Mi-2/NuRD complex protects stem cell progeny from mitogenic Notch signaling

    doi: 10.7554/eLife.41637

    Figure Lengend Snippet: Effects of Mi-2 depletion on histone modifications at the E(spl)-C locus. ( A ) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 or Hairless compared to control conditions (con: GFP RNAi). Mi-2 or Hairless knock down was performed for 3 days prior to the experiment. Note that Mi-2 and H are both downregulated indicating that the knockdown strategy was successful and E(spl)mδ, E(spl)mγ, E(spl)mα and E(spl)m7 are all significantly de-repressed in Notch-off state. ( B ) Fold change in RNA levels in Kc cells upon knockdown of Mi-2 compared to control conditions (con: GFP RNAi) and to cells with Notch activation (Nact). Note that other Notch regulated genes such as CG12290 , CG17119, Notch and regular (rgl) are de-repressed in Notch-off state and have enhanced expression in Notch-on state ( C ) Fold change in RNA levels in Kc cells upon combined knockdown of Mi-2 and Hairless compared to control conditions and single knockdown of Mi-2 or Hairless . Note that Mi-2 and H are downregulated in both single and double conditions indicating that the knockdown strategy was successful. ( D ) Enrichment of Histone H3 is indicated at E(spl)-C or regions in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). H3 distribution in Notch active and in control conditions is not altered by knockdown of Mi-2 . ( E ) Enrichment for various histone modifications such as H3K27acetylation, H3K27trimethylation and H3K56acetylation is indicated at E(spl)-C locus in Kc cells as revealed by ChIP in control (grey) or Mi-2 knockdown (for 3 days, green) conditions in Notch-off (light shading) and Notch-on (EGTA treatment 30 min; dark shading). No change in the levels of these modifications was detected following knockdown of Mi-2 . (p values: *0.01

    Article Snippet: Membranes were blocked in TBTM (TBS, 0.05% tween, 3% milk) for 1 hr prior to an overnight incubation at 4°C with primary antibodies (Rabbit anti-GFP 1:2000, Invitrogen A11122; Rabbit anti-Su(H) 1:400 Santa Cruz sc-28713; Rabbit anti-Mi-2 1:10,000 courtesy of A. Brehm; rat anti-tubulin 1:2000).

    Techniques: Activation Assay, Expressing, Chromatin Immunoprecipitation

    Prospero does not cooperate with Mi-2 in NB lineages. ( A ) Depletion of Pros enhances the number of Dpn +ve cells (green or white channels) caused by Nact over-expression, Ase marks NBs and GMCs, Pros marks progeny. ( B ) Scatter dot plot, the number of Dpn +ve cells per VNC in > Nact; > Pros Ri (N = 24, three experiments) was significantly increased compared to > Nact; > w Ri larvae (N = 27, three experiments). ( C ) Overexpression of Pros ( > YFP Pros; w–Ri) results in decreased number of Dpn +ve cells per VNC (N = 15, four experiments) compared to wild type ( > LacZ; > w Ri; N = 17, four experiments) or Mi-2 knockdown alone ( > LacZ; > Mi-2-Ri; N = 16, four experiments). Concomitant loss of Mi-2 ( > YFP Pros; > Mi-2-Ri; N = 14, four experiments) does not rescue Pros-induced NB loss, instead it further decreases the number of Dpn +ve cells. Ase marks NBs and GMCs, Mira marks NBs. ( D ) Quantification of Dpn +ve cells in each condition, where shading of symbols indicates different experimental replicates for each genotype, with mean and SEM indicated by black lines.

    Journal: eLife

    Article Title: Mi-2/NuRD complex protects stem cell progeny from mitogenic Notch signaling

    doi: 10.7554/eLife.41637

    Figure Lengend Snippet: Prospero does not cooperate with Mi-2 in NB lineages. ( A ) Depletion of Pros enhances the number of Dpn +ve cells (green or white channels) caused by Nact over-expression, Ase marks NBs and GMCs, Pros marks progeny. ( B ) Scatter dot plot, the number of Dpn +ve cells per VNC in > Nact; > Pros Ri (N = 24, three experiments) was significantly increased compared to > Nact; > w Ri larvae (N = 27, three experiments). ( C ) Overexpression of Pros ( > YFP Pros; w–Ri) results in decreased number of Dpn +ve cells per VNC (N = 15, four experiments) compared to wild type ( > LacZ; > w Ri; N = 17, four experiments) or Mi-2 knockdown alone ( > LacZ; > Mi-2-Ri; N = 16, four experiments). Concomitant loss of Mi-2 ( > YFP Pros; > Mi-2-Ri; N = 14, four experiments) does not rescue Pros-induced NB loss, instead it further decreases the number of Dpn +ve cells. Ase marks NBs and GMCs, Mira marks NBs. ( D ) Quantification of Dpn +ve cells in each condition, where shading of symbols indicates different experimental replicates for each genotype, with mean and SEM indicated by black lines.

    Article Snippet: Membranes were blocked in TBTM (TBS, 0.05% tween, 3% milk) for 1 hr prior to an overnight incubation at 4°C with primary antibodies (Rabbit anti-GFP 1:2000, Invitrogen A11122; Rabbit anti-Su(H) 1:400 Santa Cruz sc-28713; Rabbit anti-Mi-2 1:10,000 courtesy of A. Brehm; rat anti-tubulin 1:2000).

    Techniques: Over Expression

    Model summarizing the role of Mi-2 in decommissioning Notch-responsive enhancers in NB progeny. The presence of Zfh1 and Mi-2 favours the decommissioning of enhancers from E(spl) and other Notch target genes in GMCs (yellow) to ensure their expression is switched off. Notch is on in NBs (green) and off in GMCs (yellow cells) due to asymmetrical segregation of Numb (ref). GMCs divide to produce two post-mitotic neuronal cells (grey). In NB lineages with constitutive Notch activity (Nact), the presence of Mi-2 at enhancers, recruited by Zfh1 (and potentially by other factors too), is sufficient to attenuate Nact, so that E(spl) and other target genes are switched off in GMCs. In a few NB progeny the effects of Mi-2 are overcome with time, and E(spl) genes are up-regulated as they revert to NB-like cells. Depletion of Mi-2 in NB lineages expressing Nact severely compromises enhancer decommissioning so that E(spl) and other Notch target genes are upregulated in many of the GMCs. The majority of the NB progeny acquire an NB-like fate. SUPPLEMENTARY MATERIAL: Legends for videos.

    Journal: eLife

    Article Title: Mi-2/NuRD complex protects stem cell progeny from mitogenic Notch signaling

    doi: 10.7554/eLife.41637

    Figure Lengend Snippet: Model summarizing the role of Mi-2 in decommissioning Notch-responsive enhancers in NB progeny. The presence of Zfh1 and Mi-2 favours the decommissioning of enhancers from E(spl) and other Notch target genes in GMCs (yellow) to ensure their expression is switched off. Notch is on in NBs (green) and off in GMCs (yellow cells) due to asymmetrical segregation of Numb (ref). GMCs divide to produce two post-mitotic neuronal cells (grey). In NB lineages with constitutive Notch activity (Nact), the presence of Mi-2 at enhancers, recruited by Zfh1 (and potentially by other factors too), is sufficient to attenuate Nact, so that E(spl) and other target genes are switched off in GMCs. In a few NB progeny the effects of Mi-2 are overcome with time, and E(spl) genes are up-regulated as they revert to NB-like cells. Depletion of Mi-2 in NB lineages expressing Nact severely compromises enhancer decommissioning so that E(spl) and other Notch target genes are upregulated in many of the GMCs. The majority of the NB progeny acquire an NB-like fate. SUPPLEMENTARY MATERIAL: Legends for videos.

    Article Snippet: Membranes were blocked in TBTM (TBS, 0.05% tween, 3% milk) for 1 hr prior to an overnight incubation at 4°C with primary antibodies (Rabbit anti-GFP 1:2000, Invitrogen A11122; Rabbit anti-Su(H) 1:400 Santa Cruz sc-28713; Rabbit anti-Mi-2 1:10,000 courtesy of A. Brehm; rat anti-tubulin 1:2000).

    Techniques: Expressing, Activity Assay

    Mi-2 depletion enhances the activation of Notch regulated genes. ( A–B ) Depletion of Mi-2 in NB lineages that receive excessive Notch signalling results in an increase in the expression of the Notch regulated enhancers from pathetic (A; path[NRE]-GFP, green) ( A ) or CycE (B; CycE[NRE]-GFP, green) as indicated. Expression of Dpn (red) and Notch reporters (green) in wild type (control), Nact expressing ( > Nact; > w-Ri) and Nact with Mi-2 depleted ( > Nact; > Mi-2-Ri). Hyperplastic lineages with low/no (arrows) or high (arrowheads) levels of Notch reporter expression are indicated. ( C ) Grainy head (Grh) and Miranda (Mira) are upregulated when Mi-2 is depleted in NB lineages expressing Nact; Dpn +ve (green), Grh +ve (red) and Mira +ve (blue) in the indicated genotypes.

    Journal: eLife

    Article Title: Mi-2/NuRD complex protects stem cell progeny from mitogenic Notch signaling

    doi: 10.7554/eLife.41637

    Figure Lengend Snippet: Mi-2 depletion enhances the activation of Notch regulated genes. ( A–B ) Depletion of Mi-2 in NB lineages that receive excessive Notch signalling results in an increase in the expression of the Notch regulated enhancers from pathetic (A; path[NRE]-GFP, green) ( A ) or CycE (B; CycE[NRE]-GFP, green) as indicated. Expression of Dpn (red) and Notch reporters (green) in wild type (control), Nact expressing ( > Nact; > w-Ri) and Nact with Mi-2 depleted ( > Nact; > Mi-2-Ri). Hyperplastic lineages with low/no (arrows) or high (arrowheads) levels of Notch reporter expression are indicated. ( C ) Grainy head (Grh) and Miranda (Mira) are upregulated when Mi-2 is depleted in NB lineages expressing Nact; Dpn +ve (green), Grh +ve (red) and Mira +ve (blue) in the indicated genotypes.

    Article Snippet: Membranes were blocked in TBTM (TBS, 0.05% tween, 3% milk) for 1 hr prior to an overnight incubation at 4°C with primary antibodies (Rabbit anti-GFP 1:2000, Invitrogen A11122; Rabbit anti-Su(H) 1:400 Santa Cruz sc-28713; Rabbit anti-Mi-2 1:10,000 courtesy of A. Brehm; rat anti-tubulin 1:2000).

    Techniques: Activation Assay, Expressing

    Mi-2 depletion in NBs, GMCs or newly born progeny exacerbates Notch-induced tumorigenesis but Mi-2 depletion in older neurons does not. ( A ) Mi-2 knockdown leads to severe depletion of Mi-2 protein levels. Mi-2 protein (red) levels in VNCs of the indicated genotypes: Nact alone ( grhNBGal4Gal80ts > NΔecd; > w- Ri), Mi-2 knockdown in Nact ( > NΔecd; > Mi-2-Ri ) or Mi-2 knockdown alone ( > LacZ; > Mi-2-Ri ). Dpn (green) and Mira (blue) mark NBs. ( B ) Depletion of Mi-2 in NBs using insc-Gal4 leads to an increase of the number of hyperplastic Dpn +ve cells in Nact conditions. Expression of Dpn (green or white) is shown in VNCs with Nact alone ( > NΔecd; > w- Ri) and with knock down of Mi-2 in combination with Nact ( > NΔecd; > Mi-2-Ri ). Ase (red) marks NBs and GMCs, whereas Mira (blue) marks NBs ( C ) Depletion of Mi-2 in GMCs and newly born NSC progeny using cas GMR71C09 -Gal4 leads to an increase in the number of hyperplastic Dpn +ve cells in Nact conditions. Dpn (white) in wild type ( > LacZ; > w- Ri), Nact expressing ( > NΔecd; > w- Ri) knock down of Mi-2 only ( > LacZ; > Mi-2-Ri Bloomington line #33415), and knock down of Mi-2 in Nact expressing ( > NΔecd; > Mi-2-Ri ) lineages. Ase (red) marks NBs and GMCs, whereas Mira (blue) marks NBs. ( D ) Neither Nact nor Mi-2 depletion nor combination of Nact with Mi-2 depletion in mature neurons via nsyb-Gal4 lead to increase in Dpn +ve cells. Dpn (white) in knock-down of Mi-2 only ( > LacZ; > Mi-2-Ri ; Bloomington line #33415), Nact expressing ( > NΔecd; > w- Ri) and knock down of Mi-2 in Nact expressing ( > NΔecd; > Mi-2-Ri ) lineages. Ase (red) marks NBs and GMCs, whereas Mira (blue) marks NBs. ( E ). Quantification of the number of Dpn +ve cells per VNC under the conditions indicated. When Mi-2 was depleted in GMCs using cas GMR71C09 -Gal4 in Nact conditions compared to Nact alone the number of Dpn +ve cells per VNC was significantly increased. VNCs from WT or Mi-2 knockdown backgrounds exhibit normal number of Dpn +ve cells. Box represents IQR, black line indicates median and whiskers indicate ±1.5 × IQR. (*p

    Journal: eLife

    Article Title: Mi-2/NuRD complex protects stem cell progeny from mitogenic Notch signaling

    doi: 10.7554/eLife.41637

    Figure Lengend Snippet: Mi-2 depletion in NBs, GMCs or newly born progeny exacerbates Notch-induced tumorigenesis but Mi-2 depletion in older neurons does not. ( A ) Mi-2 knockdown leads to severe depletion of Mi-2 protein levels. Mi-2 protein (red) levels in VNCs of the indicated genotypes: Nact alone ( grhNBGal4Gal80ts > NΔecd; > w- Ri), Mi-2 knockdown in Nact ( > NΔecd; > Mi-2-Ri ) or Mi-2 knockdown alone ( > LacZ; > Mi-2-Ri ). Dpn (green) and Mira (blue) mark NBs. ( B ) Depletion of Mi-2 in NBs using insc-Gal4 leads to an increase of the number of hyperplastic Dpn +ve cells in Nact conditions. Expression of Dpn (green or white) is shown in VNCs with Nact alone ( > NΔecd; > w- Ri) and with knock down of Mi-2 in combination with Nact ( > NΔecd; > Mi-2-Ri ). Ase (red) marks NBs and GMCs, whereas Mira (blue) marks NBs ( C ) Depletion of Mi-2 in GMCs and newly born NSC progeny using cas GMR71C09 -Gal4 leads to an increase in the number of hyperplastic Dpn +ve cells in Nact conditions. Dpn (white) in wild type ( > LacZ; > w- Ri), Nact expressing ( > NΔecd; > w- Ri) knock down of Mi-2 only ( > LacZ; > Mi-2-Ri Bloomington line #33415), and knock down of Mi-2 in Nact expressing ( > NΔecd; > Mi-2-Ri ) lineages. Ase (red) marks NBs and GMCs, whereas Mira (blue) marks NBs. ( D ) Neither Nact nor Mi-2 depletion nor combination of Nact with Mi-2 depletion in mature neurons via nsyb-Gal4 lead to increase in Dpn +ve cells. Dpn (white) in knock-down of Mi-2 only ( > LacZ; > Mi-2-Ri ; Bloomington line #33415), Nact expressing ( > NΔecd; > w- Ri) and knock down of Mi-2 in Nact expressing ( > NΔecd; > Mi-2-Ri ) lineages. Ase (red) marks NBs and GMCs, whereas Mira (blue) marks NBs. ( E ). Quantification of the number of Dpn +ve cells per VNC under the conditions indicated. When Mi-2 was depleted in GMCs using cas GMR71C09 -Gal4 in Nact conditions compared to Nact alone the number of Dpn +ve cells per VNC was significantly increased. VNCs from WT or Mi-2 knockdown backgrounds exhibit normal number of Dpn +ve cells. Box represents IQR, black line indicates median and whiskers indicate ±1.5 × IQR. (*p

    Article Snippet: Membranes were blocked in TBTM (TBS, 0.05% tween, 3% milk) for 1 hr prior to an overnight incubation at 4°C with primary antibodies (Rabbit anti-GFP 1:2000, Invitrogen A11122; Rabbit anti-Su(H) 1:400 Santa Cruz sc-28713; Rabbit anti-Mi-2 1:10,000 courtesy of A. Brehm; rat anti-tubulin 1:2000).

    Techniques: Expressing

    Expression of Ε(spl)mγ-GFP in GMCs upon Mi-2 depletion in NB lineages. ( A ) Time points from a time lapse movie of LacZ; Mi-2-Ri NB lineages (dissociated from larval brains with grhNBGal4 Gal80ts at the permissive temperature for 24 hr). Ε(spl)mγ-GFP is switched off in emerging GMCs as in WT lineages. Upper panels, bright field images of NB and its progeny combined with Histone-RFP (red, H2Av-RFP) to monitor cell cycle stages; lower panels, expression of Ε(spl)mγ-GFP (black). Time is depicted below each panel along with cartoons of the lineages where NBs are in darker shades of green, emerging GMCs with low expression of Ε(spl)mγ-GFP in light green and GMCs with no expression of E(spl)mγ-GFP in yellow. Asterisks indicate progeny from same ancestor cell. Numbers correspond to different cells indexed in B. Scale bar 25 μm. ( B ) Bar chart depicting progression of each cell in a Mi-2-Ri NB lineage, bar thickness indicates cell-size and the colour represents Ε(spl)mγ-GFP levels according to the scale (blue low levels, yellow high levels). Dashed lines mark mitotic events linking to the emerging daughter cells. ( C ) Plot of Ε(spl)mγ-GFP levels in NBs, newly born GMCs and progeny in > LacZ; Mi-2-Ri . Note that Ε(spl)mγ-GFP levels decay in newly born GMCS in > LacZ; Mi-2-Ri lineages.

    Journal: eLife

    Article Title: Mi-2/NuRD complex protects stem cell progeny from mitogenic Notch signaling

    doi: 10.7554/eLife.41637

    Figure Lengend Snippet: Expression of Ε(spl)mγ-GFP in GMCs upon Mi-2 depletion in NB lineages. ( A ) Time points from a time lapse movie of LacZ; Mi-2-Ri NB lineages (dissociated from larval brains with grhNBGal4 Gal80ts at the permissive temperature for 24 hr). Ε(spl)mγ-GFP is switched off in emerging GMCs as in WT lineages. Upper panels, bright field images of NB and its progeny combined with Histone-RFP (red, H2Av-RFP) to monitor cell cycle stages; lower panels, expression of Ε(spl)mγ-GFP (black). Time is depicted below each panel along with cartoons of the lineages where NBs are in darker shades of green, emerging GMCs with low expression of Ε(spl)mγ-GFP in light green and GMCs with no expression of E(spl)mγ-GFP in yellow. Asterisks indicate progeny from same ancestor cell. Numbers correspond to different cells indexed in B. Scale bar 25 μm. ( B ) Bar chart depicting progression of each cell in a Mi-2-Ri NB lineage, bar thickness indicates cell-size and the colour represents Ε(spl)mγ-GFP levels according to the scale (blue low levels, yellow high levels). Dashed lines mark mitotic events linking to the emerging daughter cells. ( C ) Plot of Ε(spl)mγ-GFP levels in NBs, newly born GMCs and progeny in > LacZ; Mi-2-Ri . Note that Ε(spl)mγ-GFP levels decay in newly born GMCS in > LacZ; Mi-2-Ri lineages.

    Article Snippet: Membranes were blocked in TBTM (TBS, 0.05% tween, 3% milk) for 1 hr prior to an overnight incubation at 4°C with primary antibodies (Rabbit anti-GFP 1:2000, Invitrogen A11122; Rabbit anti-Su(H) 1:400 Santa Cruz sc-28713; Rabbit anti-Mi-2 1:10,000 courtesy of A. Brehm; rat anti-tubulin 1:2000).

    Techniques: Expressing

    Zfh1 resists Notch-induced t umorigenesis. ( A ) Ectopic expression of Zfh1 reduces the number of Dpn +ve cells (green or white channels) caused by Nact overexpression. This effect is partially rescued by the loss of Mi-2. Ase marks NBs and GMCs, Pros marks progeny. ( B ) Quantification of Dpn +ve cells per VNC in each condition, where shading of symbols indicates different experimental replicates for each genotype, mean and SEM indicated by black lines. > Nact; > zfh1 PB, > w Ri (N = 26, three experiments) was significantly decreased compared to > Nact; > LacZ, > w Ri larvae (N = 11, three experiments), whereas it remained unaltered in > Nact; > zfh1 PB, > Mi-2-Ri (N = 20, three experiments). ( C ) Mi-2-YFP and zfh1-GFP expression in type II NB lineages. Mi-2 is expressed in high levels in the Type II NB and lower levels in INPs, whereas Zfh1 is expressed only in some neurons. Yellow outlines of Type II lineages. Dpn (red) and Mira (blue) mark neuroblasts and INPs.

    Journal: eLife

    Article Title: Mi-2/NuRD complex protects stem cell progeny from mitogenic Notch signaling

    doi: 10.7554/eLife.41637

    Figure Lengend Snippet: Zfh1 resists Notch-induced t umorigenesis. ( A ) Ectopic expression of Zfh1 reduces the number of Dpn +ve cells (green or white channels) caused by Nact overexpression. This effect is partially rescued by the loss of Mi-2. Ase marks NBs and GMCs, Pros marks progeny. ( B ) Quantification of Dpn +ve cells per VNC in each condition, where shading of symbols indicates different experimental replicates for each genotype, mean and SEM indicated by black lines. > Nact; > zfh1 PB, > w Ri (N = 26, three experiments) was significantly decreased compared to > Nact; > LacZ, > w Ri larvae (N = 11, three experiments), whereas it remained unaltered in > Nact; > zfh1 PB, > Mi-2-Ri (N = 20, three experiments). ( C ) Mi-2-YFP and zfh1-GFP expression in type II NB lineages. Mi-2 is expressed in high levels in the Type II NB and lower levels in INPs, whereas Zfh1 is expressed only in some neurons. Yellow outlines of Type II lineages. Dpn (red) and Mira (blue) mark neuroblasts and INPs.

    Article Snippet: Membranes were blocked in TBTM (TBS, 0.05% tween, 3% milk) for 1 hr prior to an overnight incubation at 4°C with primary antibodies (Rabbit anti-GFP 1:2000, Invitrogen A11122; Rabbit anti-Su(H) 1:400 Santa Cruz sc-28713; Rabbit anti-Mi-2 1:10,000 courtesy of A. Brehm; rat anti-tubulin 1:2000).

    Techniques: Expressing, Over Expression