anti ehmt1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti ehmt1
    a , Immunoblotting of indicated endogenous protein levels in control and Appbp2 KO inguinal adipocytes. β-actin was used as a loading control. b , Quantification of indicated proteins in a normalized to β-actin levels. c , Global ubiquitin level in control and Appbp2 KO inguinal adipocytes. d , Relative mRNA levels of indicated genes in control and Prdm16 KO inguinal adipocytes expressing a scrambled control (Control) or shRNA targeting Appbp2 (sh- Appbp2 ). N.D., not detected. e , Upper: Oil-Red-O staining of C2C12 myoblasts expressing a scrambled control (Control) or shRNAs targeting Appbp2 . Lower: Relative Appbp2 mRNA levels in C2C12 myoblasts. f , Protein interaction between PRDM16 (aa. 881–1038) and APPBP2 in the presence and absence of <t>EHMT1.</t> g , Immunoblotting of indicated proteins in the interscapular BAT of mice at 12, 24, 48, and 74 weeks old. h , Expression of indicated proteins in the inguinal WAT and interscapular BAT of 12-weeks-old mice at 30 °C for 2 weeks, at room temperature, and at 4 °C for 24 h. Representative results in a , c, f - h from two independent experiments. Gel source data are presented in Supplementary Fig. . a – e , g , h , n = 3 per group, biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t -test ( b , d ) or one-way ANOVA followed by Dunnett’s test ( e ).
    Anti Ehmt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ehmt1/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ehmt1 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Post-translational control of beige fat biogenesis by PRDM16 stabilization"

    Article Title: Post-translational control of beige fat biogenesis by PRDM16 stabilization

    Journal: Nature

    doi: 10.1038/s41586-022-05067-4

    a , Immunoblotting of indicated endogenous protein levels in control and Appbp2 KO inguinal adipocytes. β-actin was used as a loading control. b , Quantification of indicated proteins in a normalized to β-actin levels. c , Global ubiquitin level in control and Appbp2 KO inguinal adipocytes. d , Relative mRNA levels of indicated genes in control and Prdm16 KO inguinal adipocytes expressing a scrambled control (Control) or shRNA targeting Appbp2 (sh- Appbp2 ). N.D., not detected. e , Upper: Oil-Red-O staining of C2C12 myoblasts expressing a scrambled control (Control) or shRNAs targeting Appbp2 . Lower: Relative Appbp2 mRNA levels in C2C12 myoblasts. f , Protein interaction between PRDM16 (aa. 881–1038) and APPBP2 in the presence and absence of EHMT1. g , Immunoblotting of indicated proteins in the interscapular BAT of mice at 12, 24, 48, and 74 weeks old. h , Expression of indicated proteins in the inguinal WAT and interscapular BAT of 12-weeks-old mice at 30 °C for 2 weeks, at room temperature, and at 4 °C for 24 h. Representative results in a , c, f - h from two independent experiments. Gel source data are presented in Supplementary Fig. . a – e , g , h , n = 3 per group, biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t -test ( b , d ) or one-way ANOVA followed by Dunnett’s test ( e ).
    Figure Legend Snippet: a , Immunoblotting of indicated endogenous protein levels in control and Appbp2 KO inguinal adipocytes. β-actin was used as a loading control. b , Quantification of indicated proteins in a normalized to β-actin levels. c , Global ubiquitin level in control and Appbp2 KO inguinal adipocytes. d , Relative mRNA levels of indicated genes in control and Prdm16 KO inguinal adipocytes expressing a scrambled control (Control) or shRNA targeting Appbp2 (sh- Appbp2 ). N.D., not detected. e , Upper: Oil-Red-O staining of C2C12 myoblasts expressing a scrambled control (Control) or shRNAs targeting Appbp2 . Lower: Relative Appbp2 mRNA levels in C2C12 myoblasts. f , Protein interaction between PRDM16 (aa. 881–1038) and APPBP2 in the presence and absence of EHMT1. g , Immunoblotting of indicated proteins in the interscapular BAT of mice at 12, 24, 48, and 74 weeks old. h , Expression of indicated proteins in the inguinal WAT and interscapular BAT of 12-weeks-old mice at 30 °C for 2 weeks, at room temperature, and at 4 °C for 24 h. Representative results in a , c, f - h from two independent experiments. Gel source data are presented in Supplementary Fig. . a – e , g , h , n = 3 per group, biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t -test ( b , d ) or one-way ANOVA followed by Dunnett’s test ( e ).

    Techniques Used: Western Blot, Expressing, shRNA, Staining

    a , Changes in the PRDM16–APPBP2 interaction in the presence of EHMT1. b , Changes in PRDM16 polyubiquitination in the presence of EHMT1. c , Immunoblot analysis of the indicated proteins in the inguinal WAT of mice aged 12, 24, 48 and 74 weeks. β-Actin was used as the loading control. n = 3 per group. d , The whole-body OCR of Adipo- Appbp2- KO and littermate control mice on a regular chow diet at 30 °C in response to treatment with CL-316,243. n = 8 for both groups. e , H&E staining of the inguinal WAT (anterior, middle and posterior regions) in Adipo- Appbp2- KO and control mice in d . Scale bars, 210 μm. Representative images from two biologically independent samples per group. LN, lymph node. f , The body weight of mice on an HFD. n = 10 (Adipo- Appbp2 -KO) and n = 11 (control). g , Glucose-tolerance test of mice at 9 weeks of feeding on an HFD. h , Insulin-tolerance test of mice at 10 weeks of feeding on an HFD. i , A model of how CUL2–APPBP2 controls PRDM16 protein stability and beige fat biogenesis. The CUL2–APPBP2 E3 ligase complex catalyses polyubiquitination of PRDM16. Inhibition of CUL2–APPBP2 potently extends the protein half-life of PRDM16, leading to the activation of brown/beige fat genes as well as the repression of pro-inflammatory and pro-fibrosis genes in adipocytes. CUL2–APPBP2 expression in adipose tissues increases with age, showing an inverse correlation with the age-associated decline in PRDM16 protein. EHMT1 stabilizes PRDM16 protein by blocking the PRDM16–APPBP2 interaction. Mito., mitochondrial. For a – c , representative results are shown from two independent experiments. Gel source data are presented in Supplementary Fig. . For c , d and f – h , data are from biologically independent samples. For d , f , g and h , data are mean ± s.e.m. Two-sided P values were calculated using two-way repeated-measures ANOVA ( d and f – h ) followed by Fisher’s least significant difference test ( f – h ). * P < 0.05, ** P < 0.01, *** P < 0.001. Exact P values are shown in Supplementary Table .
    Figure Legend Snippet: a , Changes in the PRDM16–APPBP2 interaction in the presence of EHMT1. b , Changes in PRDM16 polyubiquitination in the presence of EHMT1. c , Immunoblot analysis of the indicated proteins in the inguinal WAT of mice aged 12, 24, 48 and 74 weeks. β-Actin was used as the loading control. n = 3 per group. d , The whole-body OCR of Adipo- Appbp2- KO and littermate control mice on a regular chow diet at 30 °C in response to treatment with CL-316,243. n = 8 for both groups. e , H&E staining of the inguinal WAT (anterior, middle and posterior regions) in Adipo- Appbp2- KO and control mice in d . Scale bars, 210 μm. Representative images from two biologically independent samples per group. LN, lymph node. f , The body weight of mice on an HFD. n = 10 (Adipo- Appbp2 -KO) and n = 11 (control). g , Glucose-tolerance test of mice at 9 weeks of feeding on an HFD. h , Insulin-tolerance test of mice at 10 weeks of feeding on an HFD. i , A model of how CUL2–APPBP2 controls PRDM16 protein stability and beige fat biogenesis. The CUL2–APPBP2 E3 ligase complex catalyses polyubiquitination of PRDM16. Inhibition of CUL2–APPBP2 potently extends the protein half-life of PRDM16, leading to the activation of brown/beige fat genes as well as the repression of pro-inflammatory and pro-fibrosis genes in adipocytes. CUL2–APPBP2 expression in adipose tissues increases with age, showing an inverse correlation with the age-associated decline in PRDM16 protein. EHMT1 stabilizes PRDM16 protein by blocking the PRDM16–APPBP2 interaction. Mito., mitochondrial. For a – c , representative results are shown from two independent experiments. Gel source data are presented in Supplementary Fig. . For c , d and f – h , data are from biologically independent samples. For d , f , g and h , data are mean ± s.e.m. Two-sided P values were calculated using two-way repeated-measures ANOVA ( d and f – h ) followed by Fisher’s least significant difference test ( f – h ). * P < 0.05, ** P < 0.01, *** P < 0.001. Exact P values are shown in Supplementary Table .

    Techniques Used: Western Blot, Staining, Inhibition, Activation Assay, Expressing, Blocking Assay

    anti ehmt1  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc anti ehmt1
    a , Immunoblotting of indicated endogenous protein levels in control and Appbp2 KO inguinal adipocytes. β-actin was used as a loading control. b , Quantification of indicated proteins in a normalized to β-actin levels. c , Global ubiquitin level in control and Appbp2 KO inguinal adipocytes. d , Relative mRNA levels of indicated genes in control and Prdm16 KO inguinal adipocytes expressing a scrambled control (Control) or shRNA targeting Appbp2 (sh- Appbp2 ). N.D., not detected. e , Upper: Oil-Red-O staining of C2C12 myoblasts expressing a scrambled control (Control) or shRNAs targeting Appbp2 . Lower: Relative Appbp2 mRNA levels in C2C12 myoblasts. f , Protein interaction between PRDM16 (aa. 881–1038) and APPBP2 in the presence and absence of <t>EHMT1.</t> g , Immunoblotting of indicated proteins in the interscapular BAT of mice at 12, 24, 48, and 74 weeks old. h , Expression of indicated proteins in the inguinal WAT and interscapular BAT of 12-weeks-old mice at 30 °C for 2 weeks, at room temperature, and at 4 °C for 24 h. Representative results in a , c, f - h from two independent experiments. Gel source data are presented in Supplementary Fig. . a – e , g , h , n = 3 per group, biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t -test ( b , d ) or one-way ANOVA followed by Dunnett’s test ( e ).
    Anti Ehmt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ehmt1/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ehmt1 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Post-translational control of beige fat biogenesis by PRDM16 stabilization"

    Article Title: Post-translational control of beige fat biogenesis by PRDM16 stabilization

    Journal: Nature

    doi: 10.1038/s41586-022-05067-4

    a , Immunoblotting of indicated endogenous protein levels in control and Appbp2 KO inguinal adipocytes. β-actin was used as a loading control. b , Quantification of indicated proteins in a normalized to β-actin levels. c , Global ubiquitin level in control and Appbp2 KO inguinal adipocytes. d , Relative mRNA levels of indicated genes in control and Prdm16 KO inguinal adipocytes expressing a scrambled control (Control) or shRNA targeting Appbp2 (sh- Appbp2 ). N.D., not detected. e , Upper: Oil-Red-O staining of C2C12 myoblasts expressing a scrambled control (Control) or shRNAs targeting Appbp2 . Lower: Relative Appbp2 mRNA levels in C2C12 myoblasts. f , Protein interaction between PRDM16 (aa. 881–1038) and APPBP2 in the presence and absence of EHMT1. g , Immunoblotting of indicated proteins in the interscapular BAT of mice at 12, 24, 48, and 74 weeks old. h , Expression of indicated proteins in the inguinal WAT and interscapular BAT of 12-weeks-old mice at 30 °C for 2 weeks, at room temperature, and at 4 °C for 24 h. Representative results in a , c, f - h from two independent experiments. Gel source data are presented in Supplementary Fig. . a – e , g , h , n = 3 per group, biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t -test ( b , d ) or one-way ANOVA followed by Dunnett’s test ( e ).
    Figure Legend Snippet: a , Immunoblotting of indicated endogenous protein levels in control and Appbp2 KO inguinal adipocytes. β-actin was used as a loading control. b , Quantification of indicated proteins in a normalized to β-actin levels. c , Global ubiquitin level in control and Appbp2 KO inguinal adipocytes. d , Relative mRNA levels of indicated genes in control and Prdm16 KO inguinal adipocytes expressing a scrambled control (Control) or shRNA targeting Appbp2 (sh- Appbp2 ). N.D., not detected. e , Upper: Oil-Red-O staining of C2C12 myoblasts expressing a scrambled control (Control) or shRNAs targeting Appbp2 . Lower: Relative Appbp2 mRNA levels in C2C12 myoblasts. f , Protein interaction between PRDM16 (aa. 881–1038) and APPBP2 in the presence and absence of EHMT1. g , Immunoblotting of indicated proteins in the interscapular BAT of mice at 12, 24, 48, and 74 weeks old. h , Expression of indicated proteins in the inguinal WAT and interscapular BAT of 12-weeks-old mice at 30 °C for 2 weeks, at room temperature, and at 4 °C for 24 h. Representative results in a , c, f - h from two independent experiments. Gel source data are presented in Supplementary Fig. . a – e , g , h , n = 3 per group, biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t -test ( b , d ) or one-way ANOVA followed by Dunnett’s test ( e ).

    Techniques Used: Western Blot, Expressing, shRNA, Staining

    a , Changes in the PRDM16–APPBP2 interaction in the presence of EHMT1. b , Changes in PRDM16 polyubiquitination in the presence of EHMT1. c , Immunoblot analysis of the indicated proteins in the inguinal WAT of mice aged 12, 24, 48 and 74 weeks. β-Actin was used as the loading control. n = 3 per group. d , The whole-body OCR of Adipo- Appbp2- KO and littermate control mice on a regular chow diet at 30 °C in response to treatment with CL-316,243. n = 8 for both groups. e , H&E staining of the inguinal WAT (anterior, middle and posterior regions) in Adipo- Appbp2- KO and control mice in d . Scale bars, 210 μm. Representative images from two biologically independent samples per group. LN, lymph node. f , The body weight of mice on an HFD. n = 10 (Adipo- Appbp2 -KO) and n = 11 (control). g , Glucose-tolerance test of mice at 9 weeks of feeding on an HFD. h , Insulin-tolerance test of mice at 10 weeks of feeding on an HFD. i , A model of how CUL2–APPBP2 controls PRDM16 protein stability and beige fat biogenesis. The CUL2–APPBP2 E3 ligase complex catalyses polyubiquitination of PRDM16. Inhibition of CUL2–APPBP2 potently extends the protein half-life of PRDM16, leading to the activation of brown/beige fat genes as well as the repression of pro-inflammatory and pro-fibrosis genes in adipocytes. CUL2–APPBP2 expression in adipose tissues increases with age, showing an inverse correlation with the age-associated decline in PRDM16 protein. EHMT1 stabilizes PRDM16 protein by blocking the PRDM16–APPBP2 interaction. Mito., mitochondrial. For a – c , representative results are shown from two independent experiments. Gel source data are presented in Supplementary Fig. . For c , d and f – h , data are from biologically independent samples. For d , f , g and h , data are mean ± s.e.m. Two-sided P values were calculated using two-way repeated-measures ANOVA ( d and f – h ) followed by Fisher’s least significant difference test ( f – h ). * P < 0.05, ** P < 0.01, *** P < 0.001. Exact P values are shown in Supplementary Table .
    Figure Legend Snippet: a , Changes in the PRDM16–APPBP2 interaction in the presence of EHMT1. b , Changes in PRDM16 polyubiquitination in the presence of EHMT1. c , Immunoblot analysis of the indicated proteins in the inguinal WAT of mice aged 12, 24, 48 and 74 weeks. β-Actin was used as the loading control. n = 3 per group. d , The whole-body OCR of Adipo- Appbp2- KO and littermate control mice on a regular chow diet at 30 °C in response to treatment with CL-316,243. n = 8 for both groups. e , H&E staining of the inguinal WAT (anterior, middle and posterior regions) in Adipo- Appbp2- KO and control mice in d . Scale bars, 210 μm. Representative images from two biologically independent samples per group. LN, lymph node. f , The body weight of mice on an HFD. n = 10 (Adipo- Appbp2 -KO) and n = 11 (control). g , Glucose-tolerance test of mice at 9 weeks of feeding on an HFD. h , Insulin-tolerance test of mice at 10 weeks of feeding on an HFD. i , A model of how CUL2–APPBP2 controls PRDM16 protein stability and beige fat biogenesis. The CUL2–APPBP2 E3 ligase complex catalyses polyubiquitination of PRDM16. Inhibition of CUL2–APPBP2 potently extends the protein half-life of PRDM16, leading to the activation of brown/beige fat genes as well as the repression of pro-inflammatory and pro-fibrosis genes in adipocytes. CUL2–APPBP2 expression in adipose tissues increases with age, showing an inverse correlation with the age-associated decline in PRDM16 protein. EHMT1 stabilizes PRDM16 protein by blocking the PRDM16–APPBP2 interaction. Mito., mitochondrial. For a – c , representative results are shown from two independent experiments. Gel source data are presented in Supplementary Fig. . For c , d and f – h , data are from biologically independent samples. For d , f , g and h , data are mean ± s.e.m. Two-sided P values were calculated using two-way repeated-measures ANOVA ( d and f – h ) followed by Fisher’s least significant difference test ( f – h ). * P < 0.05, ** P < 0.01, *** P < 0.001. Exact P values are shown in Supplementary Table .

    Techniques Used: Western Blot, Staining, Inhibition, Activation Assay, Expressing, Blocking Assay

    rabbit anti ehmt1 antibody  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc rabbit anti ehmt1 antibody
    Confocal micrographs show the immunostained H3K27me2 (green) and DNA (DAPI, blue) in mouse zygotes. a Zygotes were cultured in KSOM medium containing 10 μM BIX01294. Control group was cultured in KSOM medium containing DMSO. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. b The H3K27me2 state in zygotes at 13 h of IVF after <t>EHMT1</t> or EHMT2 antibody microinjection. α-EHMT1 and α-EHMT2 indicate mouse zygotes cultured in KSOM medium after antibody microinjection before PN3 stage. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. c Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after treatment with 10 μM BIX01294, α-EHMT1, and α-EHMT2. Error bars, S.E.M. *** P < 1.36095E−16 and P < 1.49664E−16. n.s. represents nonsignificant difference. P > 0.4948 by two-tailed Student’s t tests. Source data are provided as a Source data profile. d The H3K27me2 state of zygotes at 13 h of IVF after EHMT1 or EHMT2 siRNA microinjection. Control group was microinjected with scrambled control siRNA. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. e Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after microinjected with Ehmt1 or Ehmt2 siRNA. Error bars, S.E.M. *** P < 7.10683E−16. n.s. represents a nonsignificant difference. P > 0.9651 by two-tailed Student’s t tests. Source data are provided as a Source data profile. f The H3K27me2 state in zygotes at 13 h of IVF after Ehmt1 or Ehmt2 mRNA microinjection. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. g Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after microinjected with Ehmt1 or Ehmt2 mRNA. Error bars, S.E.M. *** P < 5.508E-−12. n.s. represents a nonsignificant difference. P > 0.3577 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.
    Rabbit Anti Ehmt1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti ehmt1 antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti ehmt1 antibody - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "PRC2 and EHMT1 regulate H3K27me2 and H3K27me3 establishment across the zygote genome"

    Article Title: PRC2 and EHMT1 regulate H3K27me2 and H3K27me3 establishment across the zygote genome

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20242-9

    Confocal micrographs show the immunostained H3K27me2 (green) and DNA (DAPI, blue) in mouse zygotes. a Zygotes were cultured in KSOM medium containing 10 μM BIX01294. Control group was cultured in KSOM medium containing DMSO. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. b The H3K27me2 state in zygotes at 13 h of IVF after EHMT1 or EHMT2 antibody microinjection. α-EHMT1 and α-EHMT2 indicate mouse zygotes cultured in KSOM medium after antibody microinjection before PN3 stage. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. c Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after treatment with 10 μM BIX01294, α-EHMT1, and α-EHMT2. Error bars, S.E.M. *** P < 1.36095E−16 and P < 1.49664E−16. n.s. represents nonsignificant difference. P > 0.4948 by two-tailed Student’s t tests. Source data are provided as a Source data profile. d The H3K27me2 state of zygotes at 13 h of IVF after EHMT1 or EHMT2 siRNA microinjection. Control group was microinjected with scrambled control siRNA. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. e Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after microinjected with Ehmt1 or Ehmt2 siRNA. Error bars, S.E.M. *** P < 7.10683E−16. n.s. represents a nonsignificant difference. P > 0.9651 by two-tailed Student’s t tests. Source data are provided as a Source data profile. f The H3K27me2 state in zygotes at 13 h of IVF after Ehmt1 or Ehmt2 mRNA microinjection. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. g Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after microinjected with Ehmt1 or Ehmt2 mRNA. Error bars, S.E.M. *** P < 5.508E-−12. n.s. represents a nonsignificant difference. P > 0.3577 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.
    Figure Legend Snippet: Confocal micrographs show the immunostained H3K27me2 (green) and DNA (DAPI, blue) in mouse zygotes. a Zygotes were cultured in KSOM medium containing 10 μM BIX01294. Control group was cultured in KSOM medium containing DMSO. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. b The H3K27me2 state in zygotes at 13 h of IVF after EHMT1 or EHMT2 antibody microinjection. α-EHMT1 and α-EHMT2 indicate mouse zygotes cultured in KSOM medium after antibody microinjection before PN3 stage. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. c Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after treatment with 10 μM BIX01294, α-EHMT1, and α-EHMT2. Error bars, S.E.M. *** P < 1.36095E−16 and P < 1.49664E−16. n.s. represents nonsignificant difference. P > 0.4948 by two-tailed Student’s t tests. Source data are provided as a Source data profile. d The H3K27me2 state of zygotes at 13 h of IVF after EHMT1 or EHMT2 siRNA microinjection. Control group was microinjected with scrambled control siRNA. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. e Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after microinjected with Ehmt1 or Ehmt2 siRNA. Error bars, S.E.M. *** P < 7.10683E−16. n.s. represents a nonsignificant difference. P > 0.9651 by two-tailed Student’s t tests. Source data are provided as a Source data profile. f The H3K27me2 state in zygotes at 13 h of IVF after Ehmt1 or Ehmt2 mRNA microinjection. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. g Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after microinjected with Ehmt1 or Ehmt2 mRNA. Error bars, S.E.M. *** P < 5.508E-−12. n.s. represents a nonsignificant difference. P > 0.3577 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.

    Techniques Used: Cell Culture, Fluorescence, Two Tailed Test

    a Zygotes were cultured in KSOM medium containing 10 μM BIX01294. Control group was cultured in KSOM medium containing DMSO. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. b The upper figure shows the protein levels of EHMT1 in zygotes at 13 h of IVF after EHMT1 full-length ( Ehmt1 ) and SET domain deleted EHMT1 truncated mRNA ( Ehmt1 ΔSET ) microinjection, respectively. The bottom figure shows the protein levels of EHMT2 in zygotes at 13 h of IVF after EHMT2 full-length ( Ehmt2 ) mRNA microinjection. c The H3K27me3 state in zygotes at 13 h of IVF after Ehmt1 or Ehmt2 mRNA microinjection. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. d Relative fluorescence intensity of H3K27me3 in PN5 male pronucleus after treatment with BIX01294, Ehmt1, and Ehmt2 mRNA. Error bars, S.E.M. *** P < 2.10148E−16. n.s. represents a nonsignificant difference. P > 0.6153 and P > 0.7802 by two-tailed Student’s t tests. Source data are provided as a Source data profile. e The H3K27me3 state in zygotes at 13 h of IVF after Ezh2 mRNA microinjection. Ezh2-mi +BIX01294 indicates zygotes cultured in KSOM medium containing BIX01294 after ezh2 mRNA microinjection. White dashed circles indicate the male pronucleus (M) and female (F) pronucleus. Scale bar, 10 μm. f Relative fluorescence intensity of H3K27me3 in PN5 male pronucleus after different treatment strategies. Error bars, S.E.M. *** P < 1.25007E−12 and P < 1.10945E−15 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.
    Figure Legend Snippet: a Zygotes were cultured in KSOM medium containing 10 μM BIX01294. Control group was cultured in KSOM medium containing DMSO. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. b The upper figure shows the protein levels of EHMT1 in zygotes at 13 h of IVF after EHMT1 full-length ( Ehmt1 ) and SET domain deleted EHMT1 truncated mRNA ( Ehmt1 ΔSET ) microinjection, respectively. The bottom figure shows the protein levels of EHMT2 in zygotes at 13 h of IVF after EHMT2 full-length ( Ehmt2 ) mRNA microinjection. c The H3K27me3 state in zygotes at 13 h of IVF after Ehmt1 or Ehmt2 mRNA microinjection. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. d Relative fluorescence intensity of H3K27me3 in PN5 male pronucleus after treatment with BIX01294, Ehmt1, and Ehmt2 mRNA. Error bars, S.E.M. *** P < 2.10148E−16. n.s. represents a nonsignificant difference. P > 0.6153 and P > 0.7802 by two-tailed Student’s t tests. Source data are provided as a Source data profile. e The H3K27me3 state in zygotes at 13 h of IVF after Ezh2 mRNA microinjection. Ezh2-mi +BIX01294 indicates zygotes cultured in KSOM medium containing BIX01294 after ezh2 mRNA microinjection. White dashed circles indicate the male pronucleus (M) and female (F) pronucleus. Scale bar, 10 μm. f Relative fluorescence intensity of H3K27me3 in PN5 male pronucleus after different treatment strategies. Error bars, S.E.M. *** P < 1.25007E−12 and P < 1.10945E−15 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.

    Techniques Used: Cell Culture, Fluorescence, Two Tailed Test

    a Schematic representation of myc-tagged mEHMT full-length, myc-tagged mEHMT SET domain deleted protein, and myc-tagged mEHMT containing the point mutant (C1201A) in SET domain without catalytic activity. b The interaction of EHMT1 with EZH1 and EZH2 proteins were examined by Co-IP. EHMT1-HA was co-transfected into HEK293T cells with EZH1-Myc, EZH2-Myc or Myc-Vector, respectively. Anti-HA was used to precipitate EHMT1 and associated proteins. Immunoblots were probed with EHMT1 and HA antibodies to detect the pull-down proteins (EZH1, EZH2) and EHMT1-HA (positive control), respectively. c Immunostaining for the EHMT1 in zygotes at 13 h of IVF after EHMT1 full-length ( Ehmt1 ) and SET domain deleted EHMT1 truncated mRNA ( Ehmt1 ΔSET ) microinjection. DNA was counterstained with DAPI. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. d The H3K27me2 state of zygotes at 13 h of IVF after EHMT1 full-length ( Ehmt1 ) and SET domain deleted mRNA ( Ehmt1 Δ SET ) microinjection. Scale bar, 10 μm. e Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after different treatment strategies. Ehmt1 mi indicates zygotes microinjected with Ehmt1 full-length mRNA; Ehmt1 Δ SET mi indicates zygotes microinjected with Ehmt1 SET domain deleted mRNA. n = 10 zygotes at each group examined over three independent experiments. Error bars, S.E.M. *** P < 2.40369E−11and P < 1.20055E−15 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.
    Figure Legend Snippet: a Schematic representation of myc-tagged mEHMT full-length, myc-tagged mEHMT SET domain deleted protein, and myc-tagged mEHMT containing the point mutant (C1201A) in SET domain without catalytic activity. b The interaction of EHMT1 with EZH1 and EZH2 proteins were examined by Co-IP. EHMT1-HA was co-transfected into HEK293T cells with EZH1-Myc, EZH2-Myc or Myc-Vector, respectively. Anti-HA was used to precipitate EHMT1 and associated proteins. Immunoblots were probed with EHMT1 and HA antibodies to detect the pull-down proteins (EZH1, EZH2) and EHMT1-HA (positive control), respectively. c Immunostaining for the EHMT1 in zygotes at 13 h of IVF after EHMT1 full-length ( Ehmt1 ) and SET domain deleted EHMT1 truncated mRNA ( Ehmt1 ΔSET ) microinjection. DNA was counterstained with DAPI. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. d The H3K27me2 state of zygotes at 13 h of IVF after EHMT1 full-length ( Ehmt1 ) and SET domain deleted mRNA ( Ehmt1 Δ SET ) microinjection. Scale bar, 10 μm. e Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after different treatment strategies. Ehmt1 mi indicates zygotes microinjected with Ehmt1 full-length mRNA; Ehmt1 Δ SET mi indicates zygotes microinjected with Ehmt1 SET domain deleted mRNA. n = 10 zygotes at each group examined over three independent experiments. Error bars, S.E.M. *** P < 2.40369E−11and P < 1.20055E−15 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.

    Techniques Used: Mutagenesis, Activity Assay, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Western Blot, Positive Control, Immunostaining, Fluorescence, Two Tailed Test

    We propose that EHMT1 is a new contributor to the formation of H3K27me2, and H3K27me2 might be a necessary prerequisite for the re-modification of H3K27me3 in the male pronucleus. The SET domain rather than the catalytic activity of EHMT1 is indispensable for de novo H3K27me2 in mouse zygotes. In addition, EZH2 is indispensable for the formation of H3K27me3 in mouse zygotes, and EZH2 is regulated by CDK1 in a cell cycle-dependent manner. However, Ezh1 and EZH2 have a synergistic effect on the establishment of de novo H3K27me2 in the paternal pronucleus of mouse zygote.
    Figure Legend Snippet: We propose that EHMT1 is a new contributor to the formation of H3K27me2, and H3K27me2 might be a necessary prerequisite for the re-modification of H3K27me3 in the male pronucleus. The SET domain rather than the catalytic activity of EHMT1 is indispensable for de novo H3K27me2 in mouse zygotes. In addition, EZH2 is indispensable for the formation of H3K27me3 in mouse zygotes, and EZH2 is regulated by CDK1 in a cell cycle-dependent manner. However, Ezh1 and EZH2 have a synergistic effect on the establishment of de novo H3K27me2 in the paternal pronucleus of mouse zygote.

    Techniques Used: Modification, Activity Assay

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    a , Immunoblotting of indicated endogenous protein levels in control and Appbp2 KO inguinal adipocytes. β-actin was used as a loading control. b , Quantification of indicated proteins in a normalized to β-actin levels. c , Global ubiquitin level in control and Appbp2 KO inguinal adipocytes. d , Relative mRNA levels of indicated genes in control and Prdm16 KO inguinal adipocytes expressing a scrambled control (Control) or shRNA targeting Appbp2 (sh- Appbp2 ). N.D., not detected. e , Upper: Oil-Red-O staining of C2C12 myoblasts expressing a scrambled control (Control) or shRNAs targeting Appbp2 . Lower: Relative Appbp2 mRNA levels in C2C12 myoblasts. f , Protein interaction between PRDM16 (aa. 881–1038) and APPBP2 in the presence and absence of <t>EHMT1.</t> g , Immunoblotting of indicated proteins in the interscapular BAT of mice at 12, 24, 48, and 74 weeks old. h , Expression of indicated proteins in the inguinal WAT and interscapular BAT of 12-weeks-old mice at 30 °C for 2 weeks, at room temperature, and at 4 °C for 24 h. Representative results in a , c, f - h from two independent experiments. Gel source data are presented in Supplementary Fig. . a – e , g , h , n = 3 per group, biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t -test ( b , d ) or one-way ANOVA followed by Dunnett’s test ( e ).
    Anti Ehmt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti ehmt1 antibody  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc rabbit anti ehmt1 antibody
    Confocal micrographs show the immunostained H3K27me2 (green) and DNA (DAPI, blue) in mouse zygotes. a Zygotes were cultured in KSOM medium containing 10 μM BIX01294. Control group was cultured in KSOM medium containing DMSO. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. b The H3K27me2 state in zygotes at 13 h of IVF after <t>EHMT1</t> or EHMT2 antibody microinjection. α-EHMT1 and α-EHMT2 indicate mouse zygotes cultured in KSOM medium after antibody microinjection before PN3 stage. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. c Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after treatment with 10 μM BIX01294, α-EHMT1, and α-EHMT2. Error bars, S.E.M. *** P < 1.36095E−16 and P < 1.49664E−16. n.s. represents nonsignificant difference. P > 0.4948 by two-tailed Student’s t tests. Source data are provided as a Source data profile. d The H3K27me2 state of zygotes at 13 h of IVF after EHMT1 or EHMT2 siRNA microinjection. Control group was microinjected with scrambled control siRNA. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. e Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after microinjected with Ehmt1 or Ehmt2 siRNA. Error bars, S.E.M. *** P < 7.10683E−16. n.s. represents a nonsignificant difference. P > 0.9651 by two-tailed Student’s t tests. Source data are provided as a Source data profile. f The H3K27me2 state in zygotes at 13 h of IVF after Ehmt1 or Ehmt2 mRNA microinjection. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. g Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after microinjected with Ehmt1 or Ehmt2 mRNA. Error bars, S.E.M. *** P < 5.508E-−12. n.s. represents a nonsignificant difference. P > 0.3577 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.
    Rabbit Anti Ehmt1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a , Immunoblotting of indicated endogenous protein levels in control and Appbp2 KO inguinal adipocytes. β-actin was used as a loading control. b , Quantification of indicated proteins in a normalized to β-actin levels. c , Global ubiquitin level in control and Appbp2 KO inguinal adipocytes. d , Relative mRNA levels of indicated genes in control and Prdm16 KO inguinal adipocytes expressing a scrambled control (Control) or shRNA targeting Appbp2 (sh- Appbp2 ). N.D., not detected. e , Upper: Oil-Red-O staining of C2C12 myoblasts expressing a scrambled control (Control) or shRNAs targeting Appbp2 . Lower: Relative Appbp2 mRNA levels in C2C12 myoblasts. f , Protein interaction between PRDM16 (aa. 881–1038) and APPBP2 in the presence and absence of EHMT1. g , Immunoblotting of indicated proteins in the interscapular BAT of mice at 12, 24, 48, and 74 weeks old. h , Expression of indicated proteins in the inguinal WAT and interscapular BAT of 12-weeks-old mice at 30 °C for 2 weeks, at room temperature, and at 4 °C for 24 h. Representative results in a , c, f - h from two independent experiments. Gel source data are presented in Supplementary Fig. . a – e , g , h , n = 3 per group, biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t -test ( b , d ) or one-way ANOVA followed by Dunnett’s test ( e ).

    Journal: Nature

    Article Title: Post-translational control of beige fat biogenesis by PRDM16 stabilization

    doi: 10.1038/s41586-022-05067-4

    Figure Lengend Snippet: a , Immunoblotting of indicated endogenous protein levels in control and Appbp2 KO inguinal adipocytes. β-actin was used as a loading control. b , Quantification of indicated proteins in a normalized to β-actin levels. c , Global ubiquitin level in control and Appbp2 KO inguinal adipocytes. d , Relative mRNA levels of indicated genes in control and Prdm16 KO inguinal adipocytes expressing a scrambled control (Control) or shRNA targeting Appbp2 (sh- Appbp2 ). N.D., not detected. e , Upper: Oil-Red-O staining of C2C12 myoblasts expressing a scrambled control (Control) or shRNAs targeting Appbp2 . Lower: Relative Appbp2 mRNA levels in C2C12 myoblasts. f , Protein interaction between PRDM16 (aa. 881–1038) and APPBP2 in the presence and absence of EHMT1. g , Immunoblotting of indicated proteins in the interscapular BAT of mice at 12, 24, 48, and 74 weeks old. h , Expression of indicated proteins in the inguinal WAT and interscapular BAT of 12-weeks-old mice at 30 °C for 2 weeks, at room temperature, and at 4 °C for 24 h. Representative results in a , c, f - h from two independent experiments. Gel source data are presented in Supplementary Fig. . a – e , g , h , n = 3 per group, biologically independent samples. Data are mean ± s.e.m.; two-sided P values by unpaired Student’s t -test ( b , d ) or one-way ANOVA followed by Dunnett’s test ( e ).

    Article Snippet: The following antibodies were used in this study: anti-UCP1 (ab-10983, Abcam), anti-UCP1 (U6382, Sigma-Aldrich), anti-CUL2 (sc-166506, Santa Cruz), anti-recombinant CUL2 (EPR3104(2)) (ab166917, Abcam), anti-APPBP2 (NBP2-81781, Novus), anti-Flag-HRP (A8592, Sigma-Aldrich), anti-HA (sc-7392, Santa Cruz), anti-MYC (sc-40, Santa Cruz), anti-PPARγ (E-8) (sc-7273, Santa Cruz), OXPHOS cocktail (ab110413, Abcam), anti-ubiquitin (sc-8017, Santa Cruz), anti-GST (sc-138, Santa Cruz), anti-GAPDH (sc-32233, Santa Cruz), anti-β-actin (A3854, Sigma-Aldrich), anti-MUM1 (12682-1-AP, Proteintech), anti-SMAD4 (10231-1-AP, Proteintech), anti-EHD2 (11440-1-AP, Proteintech), anti-GTF2I (10499-1-AP, Proteintech), anti-HDAC1 (10197-1-AP, Proteintech), anti-CNOT1 (14276-1-AP, Proteintech), anti-CNOT9 (22503-1-AP, Proteintech), anti-CHD4 (14173-1-AP, Proteintech), anti-PRDM3 (C50E12) (2593S, Cell Signaling Technology), anti-EHMT1(E6Q8B) (35005S, Cell Signaling Technology), PhosphoPlus Akt (Ser473) Antibody Duet (8200S, Cell Signaling Technology), anti-HSP90 (4874S, Cell Signaling Technology), normal mouse IgG (sc-2025, Santa Cruz), rabbit IgG, polyclonal-isotype control (ab37415, Abcam), goat anti-rabbit light chain secondary antibody (NBP2-75935, Novus), goat anti-mouse IgG, light-chain specific antibody (91196S, Cell Signaling Technology), anti-PRDM16 (AF6295, R&D systems), polyclonal antibody against PRDM16 generated by immunizing rabbit with recombinant human PRDM16 (GenScript).

    Techniques: Western Blot, Expressing, shRNA, Staining

    a , Changes in the PRDM16–APPBP2 interaction in the presence of EHMT1. b , Changes in PRDM16 polyubiquitination in the presence of EHMT1. c , Immunoblot analysis of the indicated proteins in the inguinal WAT of mice aged 12, 24, 48 and 74 weeks. β-Actin was used as the loading control. n = 3 per group. d , The whole-body OCR of Adipo- Appbp2- KO and littermate control mice on a regular chow diet at 30 °C in response to treatment with CL-316,243. n = 8 for both groups. e , H&E staining of the inguinal WAT (anterior, middle and posterior regions) in Adipo- Appbp2- KO and control mice in d . Scale bars, 210 μm. Representative images from two biologically independent samples per group. LN, lymph node. f , The body weight of mice on an HFD. n = 10 (Adipo- Appbp2 -KO) and n = 11 (control). g , Glucose-tolerance test of mice at 9 weeks of feeding on an HFD. h , Insulin-tolerance test of mice at 10 weeks of feeding on an HFD. i , A model of how CUL2–APPBP2 controls PRDM16 protein stability and beige fat biogenesis. The CUL2–APPBP2 E3 ligase complex catalyses polyubiquitination of PRDM16. Inhibition of CUL2–APPBP2 potently extends the protein half-life of PRDM16, leading to the activation of brown/beige fat genes as well as the repression of pro-inflammatory and pro-fibrosis genes in adipocytes. CUL2–APPBP2 expression in adipose tissues increases with age, showing an inverse correlation with the age-associated decline in PRDM16 protein. EHMT1 stabilizes PRDM16 protein by blocking the PRDM16–APPBP2 interaction. Mito., mitochondrial. For a – c , representative results are shown from two independent experiments. Gel source data are presented in Supplementary Fig. . For c , d and f – h , data are from biologically independent samples. For d , f , g and h , data are mean ± s.e.m. Two-sided P values were calculated using two-way repeated-measures ANOVA ( d and f – h ) followed by Fisher’s least significant difference test ( f – h ). * P < 0.05, ** P < 0.01, *** P < 0.001. Exact P values are shown in Supplementary Table .

    Journal: Nature

    Article Title: Post-translational control of beige fat biogenesis by PRDM16 stabilization

    doi: 10.1038/s41586-022-05067-4

    Figure Lengend Snippet: a , Changes in the PRDM16–APPBP2 interaction in the presence of EHMT1. b , Changes in PRDM16 polyubiquitination in the presence of EHMT1. c , Immunoblot analysis of the indicated proteins in the inguinal WAT of mice aged 12, 24, 48 and 74 weeks. β-Actin was used as the loading control. n = 3 per group. d , The whole-body OCR of Adipo- Appbp2- KO and littermate control mice on a regular chow diet at 30 °C in response to treatment with CL-316,243. n = 8 for both groups. e , H&E staining of the inguinal WAT (anterior, middle and posterior regions) in Adipo- Appbp2- KO and control mice in d . Scale bars, 210 μm. Representative images from two biologically independent samples per group. LN, lymph node. f , The body weight of mice on an HFD. n = 10 (Adipo- Appbp2 -KO) and n = 11 (control). g , Glucose-tolerance test of mice at 9 weeks of feeding on an HFD. h , Insulin-tolerance test of mice at 10 weeks of feeding on an HFD. i , A model of how CUL2–APPBP2 controls PRDM16 protein stability and beige fat biogenesis. The CUL2–APPBP2 E3 ligase complex catalyses polyubiquitination of PRDM16. Inhibition of CUL2–APPBP2 potently extends the protein half-life of PRDM16, leading to the activation of brown/beige fat genes as well as the repression of pro-inflammatory and pro-fibrosis genes in adipocytes. CUL2–APPBP2 expression in adipose tissues increases with age, showing an inverse correlation with the age-associated decline in PRDM16 protein. EHMT1 stabilizes PRDM16 protein by blocking the PRDM16–APPBP2 interaction. Mito., mitochondrial. For a – c , representative results are shown from two independent experiments. Gel source data are presented in Supplementary Fig. . For c , d and f – h , data are from biologically independent samples. For d , f , g and h , data are mean ± s.e.m. Two-sided P values were calculated using two-way repeated-measures ANOVA ( d and f – h ) followed by Fisher’s least significant difference test ( f – h ). * P < 0.05, ** P < 0.01, *** P < 0.001. Exact P values are shown in Supplementary Table .

    Article Snippet: The following antibodies were used in this study: anti-UCP1 (ab-10983, Abcam), anti-UCP1 (U6382, Sigma-Aldrich), anti-CUL2 (sc-166506, Santa Cruz), anti-recombinant CUL2 (EPR3104(2)) (ab166917, Abcam), anti-APPBP2 (NBP2-81781, Novus), anti-Flag-HRP (A8592, Sigma-Aldrich), anti-HA (sc-7392, Santa Cruz), anti-MYC (sc-40, Santa Cruz), anti-PPARγ (E-8) (sc-7273, Santa Cruz), OXPHOS cocktail (ab110413, Abcam), anti-ubiquitin (sc-8017, Santa Cruz), anti-GST (sc-138, Santa Cruz), anti-GAPDH (sc-32233, Santa Cruz), anti-β-actin (A3854, Sigma-Aldrich), anti-MUM1 (12682-1-AP, Proteintech), anti-SMAD4 (10231-1-AP, Proteintech), anti-EHD2 (11440-1-AP, Proteintech), anti-GTF2I (10499-1-AP, Proteintech), anti-HDAC1 (10197-1-AP, Proteintech), anti-CNOT1 (14276-1-AP, Proteintech), anti-CNOT9 (22503-1-AP, Proteintech), anti-CHD4 (14173-1-AP, Proteintech), anti-PRDM3 (C50E12) (2593S, Cell Signaling Technology), anti-EHMT1(E6Q8B) (35005S, Cell Signaling Technology), PhosphoPlus Akt (Ser473) Antibody Duet (8200S, Cell Signaling Technology), anti-HSP90 (4874S, Cell Signaling Technology), normal mouse IgG (sc-2025, Santa Cruz), rabbit IgG, polyclonal-isotype control (ab37415, Abcam), goat anti-rabbit light chain secondary antibody (NBP2-75935, Novus), goat anti-mouse IgG, light-chain specific antibody (91196S, Cell Signaling Technology), anti-PRDM16 (AF6295, R&D systems), polyclonal antibody against PRDM16 generated by immunizing rabbit with recombinant human PRDM16 (GenScript).

    Techniques: Western Blot, Staining, Inhibition, Activation Assay, Expressing, Blocking Assay

    Confocal micrographs show the immunostained H3K27me2 (green) and DNA (DAPI, blue) in mouse zygotes. a Zygotes were cultured in KSOM medium containing 10 μM BIX01294. Control group was cultured in KSOM medium containing DMSO. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. b The H3K27me2 state in zygotes at 13 h of IVF after EHMT1 or EHMT2 antibody microinjection. α-EHMT1 and α-EHMT2 indicate mouse zygotes cultured in KSOM medium after antibody microinjection before PN3 stage. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. c Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after treatment with 10 μM BIX01294, α-EHMT1, and α-EHMT2. Error bars, S.E.M. *** P < 1.36095E−16 and P < 1.49664E−16. n.s. represents nonsignificant difference. P > 0.4948 by two-tailed Student’s t tests. Source data are provided as a Source data profile. d The H3K27me2 state of zygotes at 13 h of IVF after EHMT1 or EHMT2 siRNA microinjection. Control group was microinjected with scrambled control siRNA. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. e Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after microinjected with Ehmt1 or Ehmt2 siRNA. Error bars, S.E.M. *** P < 7.10683E−16. n.s. represents a nonsignificant difference. P > 0.9651 by two-tailed Student’s t tests. Source data are provided as a Source data profile. f The H3K27me2 state in zygotes at 13 h of IVF after Ehmt1 or Ehmt2 mRNA microinjection. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. g Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after microinjected with Ehmt1 or Ehmt2 mRNA. Error bars, S.E.M. *** P < 5.508E-−12. n.s. represents a nonsignificant difference. P > 0.3577 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.

    Journal: Nature Communications

    Article Title: PRC2 and EHMT1 regulate H3K27me2 and H3K27me3 establishment across the zygote genome

    doi: 10.1038/s41467-020-20242-9

    Figure Lengend Snippet: Confocal micrographs show the immunostained H3K27me2 (green) and DNA (DAPI, blue) in mouse zygotes. a Zygotes were cultured in KSOM medium containing 10 μM BIX01294. Control group was cultured in KSOM medium containing DMSO. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. b The H3K27me2 state in zygotes at 13 h of IVF after EHMT1 or EHMT2 antibody microinjection. α-EHMT1 and α-EHMT2 indicate mouse zygotes cultured in KSOM medium after antibody microinjection before PN3 stage. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. c Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after treatment with 10 μM BIX01294, α-EHMT1, and α-EHMT2. Error bars, S.E.M. *** P < 1.36095E−16 and P < 1.49664E−16. n.s. represents nonsignificant difference. P > 0.4948 by two-tailed Student’s t tests. Source data are provided as a Source data profile. d The H3K27me2 state of zygotes at 13 h of IVF after EHMT1 or EHMT2 siRNA microinjection. Control group was microinjected with scrambled control siRNA. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. e Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after microinjected with Ehmt1 or Ehmt2 siRNA. Error bars, S.E.M. *** P < 7.10683E−16. n.s. represents a nonsignificant difference. P > 0.9651 by two-tailed Student’s t tests. Source data are provided as a Source data profile. f The H3K27me2 state in zygotes at 13 h of IVF after Ehmt1 or Ehmt2 mRNA microinjection. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. g Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after microinjected with Ehmt1 or Ehmt2 mRNA. Error bars, S.E.M. *** P < 5.508E-−12. n.s. represents a nonsignificant difference. P > 0.3577 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.

    Article Snippet: The following primary antibodies were used, respectively: Mouse anti-Ezh2 antibody (BD Bioscience; 1:200), rabbit anti-Ezh1 antibody (Abcam; ab176115), rabbit anti-EHMT1 antibody (Abcam; ab41969), rabbit anti-H3K27me2 antibody (for IF, Cell Signaling, 9728S), another rabbit anti-H3K27me2 antibody (for IF, Mybiosource, MBS126234), rabbit anti-H3K27me3 antibody (for IF, Cell Signaling, 9733S), antibodies were diluted by 1% BSA/PBS. anti-H3K27me2 (for ULI-NChIP, Actif Motif; 61435).

    Techniques: Cell Culture, Fluorescence, Two Tailed Test

    a Zygotes were cultured in KSOM medium containing 10 μM BIX01294. Control group was cultured in KSOM medium containing DMSO. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. b The upper figure shows the protein levels of EHMT1 in zygotes at 13 h of IVF after EHMT1 full-length ( Ehmt1 ) and SET domain deleted EHMT1 truncated mRNA ( Ehmt1 ΔSET ) microinjection, respectively. The bottom figure shows the protein levels of EHMT2 in zygotes at 13 h of IVF after EHMT2 full-length ( Ehmt2 ) mRNA microinjection. c The H3K27me3 state in zygotes at 13 h of IVF after Ehmt1 or Ehmt2 mRNA microinjection. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. d Relative fluorescence intensity of H3K27me3 in PN5 male pronucleus after treatment with BIX01294, Ehmt1, and Ehmt2 mRNA. Error bars, S.E.M. *** P < 2.10148E−16. n.s. represents a nonsignificant difference. P > 0.6153 and P > 0.7802 by two-tailed Student’s t tests. Source data are provided as a Source data profile. e The H3K27me3 state in zygotes at 13 h of IVF after Ezh2 mRNA microinjection. Ezh2-mi +BIX01294 indicates zygotes cultured in KSOM medium containing BIX01294 after ezh2 mRNA microinjection. White dashed circles indicate the male pronucleus (M) and female (F) pronucleus. Scale bar, 10 μm. f Relative fluorescence intensity of H3K27me3 in PN5 male pronucleus after different treatment strategies. Error bars, S.E.M. *** P < 1.25007E−12 and P < 1.10945E−15 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.

    Journal: Nature Communications

    Article Title: PRC2 and EHMT1 regulate H3K27me2 and H3K27me3 establishment across the zygote genome

    doi: 10.1038/s41467-020-20242-9

    Figure Lengend Snippet: a Zygotes were cultured in KSOM medium containing 10 μM BIX01294. Control group was cultured in KSOM medium containing DMSO. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. b The upper figure shows the protein levels of EHMT1 in zygotes at 13 h of IVF after EHMT1 full-length ( Ehmt1 ) and SET domain deleted EHMT1 truncated mRNA ( Ehmt1 ΔSET ) microinjection, respectively. The bottom figure shows the protein levels of EHMT2 in zygotes at 13 h of IVF after EHMT2 full-length ( Ehmt2 ) mRNA microinjection. c The H3K27me3 state in zygotes at 13 h of IVF after Ehmt1 or Ehmt2 mRNA microinjection. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. d Relative fluorescence intensity of H3K27me3 in PN5 male pronucleus after treatment with BIX01294, Ehmt1, and Ehmt2 mRNA. Error bars, S.E.M. *** P < 2.10148E−16. n.s. represents a nonsignificant difference. P > 0.6153 and P > 0.7802 by two-tailed Student’s t tests. Source data are provided as a Source data profile. e The H3K27me3 state in zygotes at 13 h of IVF after Ezh2 mRNA microinjection. Ezh2-mi +BIX01294 indicates zygotes cultured in KSOM medium containing BIX01294 after ezh2 mRNA microinjection. White dashed circles indicate the male pronucleus (M) and female (F) pronucleus. Scale bar, 10 μm. f Relative fluorescence intensity of H3K27me3 in PN5 male pronucleus after different treatment strategies. Error bars, S.E.M. *** P < 1.25007E−12 and P < 1.10945E−15 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.

    Article Snippet: The following primary antibodies were used, respectively: Mouse anti-Ezh2 antibody (BD Bioscience; 1:200), rabbit anti-Ezh1 antibody (Abcam; ab176115), rabbit anti-EHMT1 antibody (Abcam; ab41969), rabbit anti-H3K27me2 antibody (for IF, Cell Signaling, 9728S), another rabbit anti-H3K27me2 antibody (for IF, Mybiosource, MBS126234), rabbit anti-H3K27me3 antibody (for IF, Cell Signaling, 9733S), antibodies were diluted by 1% BSA/PBS. anti-H3K27me2 (for ULI-NChIP, Actif Motif; 61435).

    Techniques: Cell Culture, Fluorescence, Two Tailed Test

    a Schematic representation of myc-tagged mEHMT full-length, myc-tagged mEHMT SET domain deleted protein, and myc-tagged mEHMT containing the point mutant (C1201A) in SET domain without catalytic activity. b The interaction of EHMT1 with EZH1 and EZH2 proteins were examined by Co-IP. EHMT1-HA was co-transfected into HEK293T cells with EZH1-Myc, EZH2-Myc or Myc-Vector, respectively. Anti-HA was used to precipitate EHMT1 and associated proteins. Immunoblots were probed with EHMT1 and HA antibodies to detect the pull-down proteins (EZH1, EZH2) and EHMT1-HA (positive control), respectively. c Immunostaining for the EHMT1 in zygotes at 13 h of IVF after EHMT1 full-length ( Ehmt1 ) and SET domain deleted EHMT1 truncated mRNA ( Ehmt1 ΔSET ) microinjection. DNA was counterstained with DAPI. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. d The H3K27me2 state of zygotes at 13 h of IVF after EHMT1 full-length ( Ehmt1 ) and SET domain deleted mRNA ( Ehmt1 Δ SET ) microinjection. Scale bar, 10 μm. e Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after different treatment strategies. Ehmt1 mi indicates zygotes microinjected with Ehmt1 full-length mRNA; Ehmt1 Δ SET mi indicates zygotes microinjected with Ehmt1 SET domain deleted mRNA. n = 10 zygotes at each group examined over three independent experiments. Error bars, S.E.M. *** P < 2.40369E−11and P < 1.20055E−15 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.

    Journal: Nature Communications

    Article Title: PRC2 and EHMT1 regulate H3K27me2 and H3K27me3 establishment across the zygote genome

    doi: 10.1038/s41467-020-20242-9

    Figure Lengend Snippet: a Schematic representation of myc-tagged mEHMT full-length, myc-tagged mEHMT SET domain deleted protein, and myc-tagged mEHMT containing the point mutant (C1201A) in SET domain without catalytic activity. b The interaction of EHMT1 with EZH1 and EZH2 proteins were examined by Co-IP. EHMT1-HA was co-transfected into HEK293T cells with EZH1-Myc, EZH2-Myc or Myc-Vector, respectively. Anti-HA was used to precipitate EHMT1 and associated proteins. Immunoblots were probed with EHMT1 and HA antibodies to detect the pull-down proteins (EZH1, EZH2) and EHMT1-HA (positive control), respectively. c Immunostaining for the EHMT1 in zygotes at 13 h of IVF after EHMT1 full-length ( Ehmt1 ) and SET domain deleted EHMT1 truncated mRNA ( Ehmt1 ΔSET ) microinjection. DNA was counterstained with DAPI. M indicates male pronucleus and F indicates female pronucleus in zygote. Scale bar, 10 μm. d The H3K27me2 state of zygotes at 13 h of IVF after EHMT1 full-length ( Ehmt1 ) and SET domain deleted mRNA ( Ehmt1 Δ SET ) microinjection. Scale bar, 10 μm. e Relative fluorescence intensity of H3K27me2 in PN5 male pronucleus after different treatment strategies. Ehmt1 mi indicates zygotes microinjected with Ehmt1 full-length mRNA; Ehmt1 Δ SET mi indicates zygotes microinjected with Ehmt1 SET domain deleted mRNA. n = 10 zygotes at each group examined over three independent experiments. Error bars, S.E.M. *** P < 2.40369E−11and P < 1.20055E−15 by two-tailed Student’s t tests. Source data are provided as a Source data profile. The median line of the box plot represents the median, and the top and bottom of the box represent the upper and lower quartile, respectively.

    Article Snippet: The following primary antibodies were used, respectively: Mouse anti-Ezh2 antibody (BD Bioscience; 1:200), rabbit anti-Ezh1 antibody (Abcam; ab176115), rabbit anti-EHMT1 antibody (Abcam; ab41969), rabbit anti-H3K27me2 antibody (for IF, Cell Signaling, 9728S), another rabbit anti-H3K27me2 antibody (for IF, Mybiosource, MBS126234), rabbit anti-H3K27me3 antibody (for IF, Cell Signaling, 9733S), antibodies were diluted by 1% BSA/PBS. anti-H3K27me2 (for ULI-NChIP, Actif Motif; 61435).

    Techniques: Mutagenesis, Activity Assay, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Western Blot, Positive Control, Immunostaining, Fluorescence, Two Tailed Test

    We propose that EHMT1 is a new contributor to the formation of H3K27me2, and H3K27me2 might be a necessary prerequisite for the re-modification of H3K27me3 in the male pronucleus. The SET domain rather than the catalytic activity of EHMT1 is indispensable for de novo H3K27me2 in mouse zygotes. In addition, EZH2 is indispensable for the formation of H3K27me3 in mouse zygotes, and EZH2 is regulated by CDK1 in a cell cycle-dependent manner. However, Ezh1 and EZH2 have a synergistic effect on the establishment of de novo H3K27me2 in the paternal pronucleus of mouse zygote.

    Journal: Nature Communications

    Article Title: PRC2 and EHMT1 regulate H3K27me2 and H3K27me3 establishment across the zygote genome

    doi: 10.1038/s41467-020-20242-9

    Figure Lengend Snippet: We propose that EHMT1 is a new contributor to the formation of H3K27me2, and H3K27me2 might be a necessary prerequisite for the re-modification of H3K27me3 in the male pronucleus. The SET domain rather than the catalytic activity of EHMT1 is indispensable for de novo H3K27me2 in mouse zygotes. In addition, EZH2 is indispensable for the formation of H3K27me3 in mouse zygotes, and EZH2 is regulated by CDK1 in a cell cycle-dependent manner. However, Ezh1 and EZH2 have a synergistic effect on the establishment of de novo H3K27me2 in the paternal pronucleus of mouse zygote.

    Article Snippet: The following primary antibodies were used, respectively: Mouse anti-Ezh2 antibody (BD Bioscience; 1:200), rabbit anti-Ezh1 antibody (Abcam; ab176115), rabbit anti-EHMT1 antibody (Abcam; ab41969), rabbit anti-H3K27me2 antibody (for IF, Cell Signaling, 9728S), another rabbit anti-H3K27me2 antibody (for IF, Mybiosource, MBS126234), rabbit anti-H3K27me3 antibody (for IF, Cell Signaling, 9733S), antibodies were diluted by 1% BSA/PBS. anti-H3K27me2 (for ULI-NChIP, Actif Motif; 61435).

    Techniques: Modification, Activity Assay