anti mis  (Santa Cruz Biotechnology)

 
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    Name:
    MIS Antibody
    Description:
    Anti MIS Antibody B 11 is a mouse monoclonal IgG1 kappa light chain MIS antibody provided at 200 µg ml specific for an epitope mapping between amino acids 541 560 at the C terminus of MIS of human origin Anti MIS Antibody B 11 is recommended for detection of precursor and mature MIS of mouse rat and human origin by WB IP IF and ELISA also reactive with additional species including and equine canine bovine and porcine Anti MIS Antibody B 11 is available conjugated to agarose for IP HRP for WB IHC P and ELISA and to either phycoerythrin or FITC for IF IHC P and FCM also available conjugated to Alexa Fluor 488 Alexa Fluor 546 Alexa Fluor 594 or Alexa Fluor 647 for WB RGB IF IHC P and FCM and for use with RGB fluorescent imaging systems such as iBright FL1000 FluorChem Typhoon Azure and other comparable systems also available conjugated to Alexa Fluor 680 or Alexa Fluor 790 for WB NIR IF and FCM for use with Near Infrared NIR detection systems such as LI COR Odyssey iBright FL1000 FluorChem Typhoon Azure and other comparable systems blocking peptide sc 166752 P Contact our Technical Service Department or your local Distributor for more information on how to receive a FREE 10 µg sample of MIS B 11 sc 166752
    Catalog Number:
    SC-166752
    Price:
    None
    Category:
    Antibodies Primary Antibodies and ImmunoCruz Conjugates Growth Factors and Hormones MIS Antibodies MIS Antibody B 11
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    Structured Review

    Santa Cruz Biotechnology anti mis
    Anti MIS Antibody B 11 is a mouse monoclonal IgG1 kappa light chain MIS antibody provided at 200 µg ml specific for an epitope mapping between amino acids 541 560 at the C terminus of MIS of human origin Anti MIS Antibody B 11 is recommended for detection of precursor and mature MIS of mouse rat and human origin by WB IP IF and ELISA also reactive with additional species including and equine canine bovine and porcine Anti MIS Antibody B 11 is available conjugated to agarose for IP HRP for WB IHC P and ELISA and to either phycoerythrin or FITC for IF IHC P and FCM also available conjugated to Alexa Fluor 488 Alexa Fluor 546 Alexa Fluor 594 or Alexa Fluor 647 for WB RGB IF IHC P and FCM and for use with RGB fluorescent imaging systems such as iBright FL1000 FluorChem Typhoon Azure and other comparable systems also available conjugated to Alexa Fluor 680 or Alexa Fluor 790 for WB NIR IF and FCM for use with Near Infrared NIR detection systems such as LI COR Odyssey iBright FL1000 FluorChem Typhoon Azure and other comparable systems blocking peptide sc 166752 P Contact our Technical Service Department or your local Distributor for more information on how to receive a FREE 10 µg sample of MIS B 11 sc 166752
    https://www.bioz.com/result/anti mis/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti mis - by Bioz Stars, 2021-07
    86/100 stars

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    Related Articles

    Incubation:

    Article Title: Effects of Gestational Diethylstilbestrol Treatment on Male and Female Gonads during Early Embryonic Development
    Article Snippet: Endogenous peroxidase activity was blocked by immersion in 3% (vol/vol) hydrogen peroxide in PBS for 15 min, and the slides were washed three times in PBS. .. The sections were incubated in a blocking solution, 5% Block Ace (Dainippon Pharmaceutical Co., Ltd., Tokyo, Japan), at room temperature for 30 min and subsequently incubated with a diluted primary antibody overnight at 4 C. Rabbit anti-SF-1 antibody (17; 1:5000 dilution), rabbit anti-P450scc antibody (AB1244, Chemicon International, Inc., Temecula, CA; 1:5000 dilution), goat anti-MIS antibody (sc-6886; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:5000 dilution), and rabbit anti-Sox9 antibody (sc-20095; Santa Cruz; 1:500 dilution) were used as primary antibodies. .. After washing with PBS +0.1% Triton X-100, sections were incubated with either N -histofine simple stain mouse MAX PO (R) (Nichirei Biosciences, Tokyo, Japan), N -histofine simple stain mouse MAX PO (G) (Nichirei), or N -histofine mouse stain (Nichirei), followed by staining using the VectaDAB substrate kit (Vector Laboratories, Burlingame, CA).

    Article Title: Photoperiod-dependent modulation of anti-Müllerian hormone in female Siberian hamsters, Phodopus sungorus
    Article Snippet: Sections were then incubated in 10% normal rabbit serum diluted in dilution buffer (0.5M Sodium chloride, 0.01M Phosphate Buffer, 3% BSA, 0.3% Triton-X 100) for 20 min at room temperature to block nonspecific binding sites. .. Polyclonal goat anti-MIS antibody (sc-6886, Santa Cruz Biotechnology, Santa Cruz, CA) was diluted 1:1000 in dilution buffer and incubated with sections for 16 hours at 4°C. .. As a negative control, primary antibody was incubated 1:1 overnight with MIS (C-20) Blocking Peptide (sc-6886P, Santa Cruz Biotechnology, Santa Cruz, CA) on a rocker at 4°C before its application to sections as described above.

    Article Title: Effects of GnRH agonists on the expression of developmental follicular anti-mullerian hormone in varying follicular stages in cyclic mice in vivo
    Article Snippet: Following dewaxing in xylene, rehydration in a series of ethanols and antigen retrieval in a microwave oven (15 min), endogenous peroxidase activity was blocked with 3% H2 O2 for 15 min. To block non-specific binding sites, the sections were then incubated in 10% normal donkey serum diluted in 0.01 M phosphate-buffered saline (PBS) for 15 min. .. The sections were incubated at 4°C overnight with polyclonal goat anti-MIS antibody (cat. no. sc-6886, Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted 1:50 in 0.01 M PBS. .. The sections were then incubated for 1 h with secondary horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG-HRP (cat. no sc-2020; Santa Cruz Biotechnology), diluted 1:100 in 0.01 M PBS.

    Blocking Assay:

    Article Title: Effects of Gestational Diethylstilbestrol Treatment on Male and Female Gonads during Early Embryonic Development
    Article Snippet: Endogenous peroxidase activity was blocked by immersion in 3% (vol/vol) hydrogen peroxide in PBS for 15 min, and the slides were washed three times in PBS. .. The sections were incubated in a blocking solution, 5% Block Ace (Dainippon Pharmaceutical Co., Ltd., Tokyo, Japan), at room temperature for 30 min and subsequently incubated with a diluted primary antibody overnight at 4 C. Rabbit anti-SF-1 antibody (17; 1:5000 dilution), rabbit anti-P450scc antibody (AB1244, Chemicon International, Inc., Temecula, CA; 1:5000 dilution), goat anti-MIS antibody (sc-6886; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:5000 dilution), and rabbit anti-Sox9 antibody (sc-20095; Santa Cruz; 1:500 dilution) were used as primary antibodies. .. After washing with PBS +0.1% Triton X-100, sections were incubated with either N -histofine simple stain mouse MAX PO (R) (Nichirei Biosciences, Tokyo, Japan), N -histofine simple stain mouse MAX PO (G) (Nichirei), or N -histofine mouse stain (Nichirei), followed by staining using the VectaDAB substrate kit (Vector Laboratories, Burlingame, CA).

    Mouse Assay:

    Article Title: ErbB4, a Receptor Tyrosine Kinase, Coordinates Organization of the Seminiferous Tubules in the Developing Testis
    Article Snippet: The nuclei were depicted with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). .. Testes were prepared from embryonic and adult mice, and the proteins were purified immediately and subjected to Western blotting using anti-ErbB4 (E200; Abcam), anti-Akt (sc-1618; Santa Cruz Biotechnology), anti-phospho-Akt (9271; Cell Signaling Technology), anti-Erk (9102; Cell Signaling Technology), anti-phospho-Erk (9101; Cell Signaling Technology), anti-MIS (sc-28912; Santa Cruz Biotechnology), or anti-Actin (sc-1616; Santa Cruz Biotechnology) antibodies as described previously ( ). .. To analyze possible changes in sperm motility due to Erbb4 knockout, cauda epididymides were dissected from 6-month-old Erbb4Flox/Flox ;MIsCre + and WT mice (n = 5 for each genotype) and transferred to 500 μL of KSOM Embryo Culture Medium (Speciality Media; Millipore).

    Purification:

    Article Title: ErbB4, a Receptor Tyrosine Kinase, Coordinates Organization of the Seminiferous Tubules in the Developing Testis
    Article Snippet: The nuclei were depicted with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). .. Testes were prepared from embryonic and adult mice, and the proteins were purified immediately and subjected to Western blotting using anti-ErbB4 (E200; Abcam), anti-Akt (sc-1618; Santa Cruz Biotechnology), anti-phospho-Akt (9271; Cell Signaling Technology), anti-Erk (9102; Cell Signaling Technology), anti-phospho-Erk (9101; Cell Signaling Technology), anti-MIS (sc-28912; Santa Cruz Biotechnology), or anti-Actin (sc-1616; Santa Cruz Biotechnology) antibodies as described previously ( ). .. To analyze possible changes in sperm motility due to Erbb4 knockout, cauda epididymides were dissected from 6-month-old Erbb4Flox/Flox ;MIsCre + and WT mice (n = 5 for each genotype) and transferred to 500 μL of KSOM Embryo Culture Medium (Speciality Media; Millipore).

    Western Blot:

    Article Title: ErbB4, a Receptor Tyrosine Kinase, Coordinates Organization of the Seminiferous Tubules in the Developing Testis
    Article Snippet: The nuclei were depicted with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). .. Testes were prepared from embryonic and adult mice, and the proteins were purified immediately and subjected to Western blotting using anti-ErbB4 (E200; Abcam), anti-Akt (sc-1618; Santa Cruz Biotechnology), anti-phospho-Akt (9271; Cell Signaling Technology), anti-Erk (9102; Cell Signaling Technology), anti-phospho-Erk (9101; Cell Signaling Technology), anti-MIS (sc-28912; Santa Cruz Biotechnology), or anti-Actin (sc-1616; Santa Cruz Biotechnology) antibodies as described previously ( ). .. To analyze possible changes in sperm motility due to Erbb4 knockout, cauda epididymides were dissected from 6-month-old Erbb4Flox/Flox ;MIsCre + and WT mice (n = 5 for each genotype) and transferred to 500 μL of KSOM Embryo Culture Medium (Speciality Media; Millipore).

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    Santa Cruz Biotechnology goat anti mis antibody
    Localization of <t>MIS,</t> Sox9, and <t>P450scc</t> proteins in DES-treated testis. IHC for MIS, Sox9, and P450scc proteins was performed on sections of fetal mouse testis at E12.5 (MIS) and E14.5 (MIS, Sox9, and P450scc) from mice exposed in utero to vehicle ( left panels ) or DES ( right panels ) (original magnification, ×132).
    Goat Anti Mis Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti mis antibody/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti mis antibody - by Bioz Stars, 2021-07
    86/100 stars
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    92
    Santa Cruz Biotechnology anti mi 2β mab
    The Mi-2/NuRD repression complex is recruited to the Il12b promoter region to regulate its transcription by lincRNA-Cox2. A, B ) Effects of lincRNA-Cox2 knockdown on the recruitment of NF-ĸB p65 and <t>Mi-2β</t> to Il12b promoter induced by TNF-α.
    Anti Mi 2β Mab, 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
    https://www.bioz.com/result/anti mi 2β mab/product/Santa Cruz Biotechnology
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti mi 2β mab - by Bioz Stars, 2021-07
    92/100 stars
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    92
    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
    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, 2021-07
    92/100 stars
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    Image Search Results


    Localization of MIS, Sox9, and P450scc proteins in DES-treated testis. IHC for MIS, Sox9, and P450scc proteins was performed on sections of fetal mouse testis at E12.5 (MIS) and E14.5 (MIS, Sox9, and P450scc) from mice exposed in utero to vehicle ( left panels ) or DES ( right panels ) (original magnification, ×132).

    Journal: Endocrinology

    Article Title: Effects of Gestational Diethylstilbestrol Treatment on Male and Female Gonads during Early Embryonic Development

    doi: 10.1210/en.2007-1599

    Figure Lengend Snippet: Localization of MIS, Sox9, and P450scc proteins in DES-treated testis. IHC for MIS, Sox9, and P450scc proteins was performed on sections of fetal mouse testis at E12.5 (MIS) and E14.5 (MIS, Sox9, and P450scc) from mice exposed in utero to vehicle ( left panels ) or DES ( right panels ) (original magnification, ×132).

    Article Snippet: The sections were incubated in a blocking solution, 5% Block Ace (Dainippon Pharmaceutical Co., Ltd., Tokyo, Japan), at room temperature for 30 min and subsequently incubated with a diluted primary antibody overnight at 4 C. Rabbit anti-SF-1 antibody (17; 1:5000 dilution), rabbit anti-P450scc antibody (AB1244, Chemicon International, Inc., Temecula, CA; 1:5000 dilution), goat anti-MIS antibody (sc-6886; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:5000 dilution), and rabbit anti-Sox9 antibody (sc-20095; Santa Cruz; 1:500 dilution) were used as primary antibodies.

    Techniques: Immunohistochemistry, Mouse Assay, In Utero

    Localization of mRNAs of SF-1, MIS, and P450scc in DES-treated testis. In situ hybridization using 35 S-labeled cRNA probe was performed to analyze localization of mRNAs of SF-1, MIS, and P450scc on sections of fetal mouse testis at E14.5. Left and right panels show testis from mice treated in utero with vehicle and DES, respectively (original magnification, ×33).

    Journal: Endocrinology

    Article Title: Effects of Gestational Diethylstilbestrol Treatment on Male and Female Gonads during Early Embryonic Development

    doi: 10.1210/en.2007-1599

    Figure Lengend Snippet: Localization of mRNAs of SF-1, MIS, and P450scc in DES-treated testis. In situ hybridization using 35 S-labeled cRNA probe was performed to analyze localization of mRNAs of SF-1, MIS, and P450scc on sections of fetal mouse testis at E14.5. Left and right panels show testis from mice treated in utero with vehicle and DES, respectively (original magnification, ×33).

    Article Snippet: The sections were incubated in a blocking solution, 5% Block Ace (Dainippon Pharmaceutical Co., Ltd., Tokyo, Japan), at room temperature for 30 min and subsequently incubated with a diluted primary antibody overnight at 4 C. Rabbit anti-SF-1 antibody (17; 1:5000 dilution), rabbit anti-P450scc antibody (AB1244, Chemicon International, Inc., Temecula, CA; 1:5000 dilution), goat anti-MIS antibody (sc-6886; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:5000 dilution), and rabbit anti-Sox9 antibody (sc-20095; Santa Cruz; 1:500 dilution) were used as primary antibodies.

    Techniques: In Situ Hybridization, Labeling, Mouse Assay, In Utero

    Design of new recombinant MIS constructs with the albumin leader sequence. ( a ) The leader sequence of MIS (25AA) and albumin (24AA) have 20% identity and 5 conserved AA. ( b ) The design of the RF, LRF, and LR constructs including the placement of the flag

    Journal: Technology (Elmsford, N.Y.)

    Article Title: An albumin leader sequence coupled with a cleavage site modification enhances the yield of recombinant C-terminal Mullerian Inhibiting Substance

    doi: 10.1142/S2339547813500076

    Figure Lengend Snippet: Design of new recombinant MIS constructs with the albumin leader sequence. ( a ) The leader sequence of MIS (25AA) and albumin (24AA) have 20% identity and 5 conserved AA. ( b ) The design of the RF, LRF, and LR constructs including the placement of the flag

    Article Snippet: Membranes were blocked with 1x PBS, 0.1% Tween-20 containing 5% nonfat dry milk for 30 minutes at room temperature and probed with horseradish peroxidase conjugated mouse monoclonal anti-FLAG M2 antibody (SIGMA) (1:1000), goat C20 anti-holo and C-terminus MIS antibody (Santa Cruz) (1:200), or rabbit MGH4 anti-holo and N-terminus MIS antibody (custom) (1:1000).

    Techniques: Recombinant, Construct, Sequencing

    The Mi-2/NuRD repression complex is recruited to the Il12b promoter region to regulate its transcription by lincRNA-Cox2. A, B ) Effects of lincRNA-Cox2 knockdown on the recruitment of NF-ĸB p65 and Mi-2β to Il12b promoter induced by TNF-α.

    Journal: The FASEB Journal

    Article Title: LincRNA-Cox2 modulates TNF-α–induced transcription of Il12b gene in intestinal epithelial cells through regulation of Mi-2/NuRD-mediated epigenetic histone modifications

    doi: 10.1096/fj.15-279166

    Figure Lengend Snippet: The Mi-2/NuRD repression complex is recruited to the Il12b promoter region to regulate its transcription by lincRNA-Cox2. A, B ) Effects of lincRNA-Cox2 knockdown on the recruitment of NF-ĸB p65 and Mi-2β to Il12b promoter induced by TNF-α.

    Article Snippet: One percent of the cell extracts was taken as input, and the rest of the extracts was incubated with either anti-p65 mAb (Santa Cruz Biotechnology), anti-Mi-2β mAb (Santa Cruz Biotechnology), anti-acetylation of histone H3 lysine 9 (H3K9ac; Abcam, Cambridge, MA, USA), anti-acetylation of histone H3 lysine 27 (H3K27ac; Abcam), anti-dimethylation of histone H3 lysine 9 (H3K9me2), anti-dimethylation of histone H3 lysine 27 (H3K27me2; Cell Signaling Technologies), anti-trimethylation of histone H3 lysine 4 (H3K4me3; Cell Signaling Technologies), anti-trimethylation of histone H3 lysine 9 (H3K9me3; Upstate Biotechnology, Waltham, MA, USA), and anti-trimethylation of histone H3 lysine 27 (H3K27me3; Cell Signaling Technologies), anti-H3K4me3 Ab (Cell Signaling Technologies), anti-H3K9me3 Ab (Upstate Biotechnology, Waltham MA, USA), anti-H3K27me3 Ab (Cell Signaling Technologies), or control IgG overnight at 4°C, followed by precipitation with protein G-agarose beads.

    Techniques:

    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