rabbit polyclonal anti ezh2 antibody  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: Gestational diabetes mellitus causes genome hyper-methylation of oocyte via increased EZH2

doi: 10.1038/s41467-024-55499-x

Figure Lengend Snippet: a differentially expressed genes (DEGs) analysis in F1 oocytes; b the association between DMRs located at promoter regions and DEGs; blue, downregulated DEGs; red, upregulated DEGs; c the mRNA expression of DNMT3a (p = 0.16794) and DNMT3l (p = 0.27292) in GDF1 oocytes; data presented as mean ± SD; d , e the mRNA expression of Ezh2 (p = 0.004) and Suz12 (p = 0.014) in GDF1 oocytes examined using qPCR; data presented as mean ± SD; f H3K27me3 modification in GV oocytes is examined using immunofluorescence and g the relative intensity of fluorescence is calculated using Image J; data presented as mean ± SD; NGDF1, n = 51; GDF1, n = 40; p = 0.01135; h the expression of EZH2 (n = 59) during folliculogenesis is examined using immunofluorescence histochemistry, and i the relative intensity fluorescence is calculated using Image J; data presented as mean ± SEM; n: primordial follicles = 28, primary follicles = 32, secondary follicles = 46, and antral follicles = 20 (p = 1.1527*10 −8 ); 8 ovaries from 8 mice were used; j the H3K27me3 (n = 36) modification in follicular development is examined, and k the relative intensity of fluorescence was calculated by Image J; data presented as mean ± SEM; n: primordial follicles=27, primary follicles = 38 (p = 1.0952*10 −12 ), secondary follicles = 33, and antral follicles = 19. *p < 0.05; **p < 0.01. Bar, 20 μm. FPKM, fragments Per Kilobase of exon model per Million mapped fragments. Source data are provided as a Source Data file. The statistical difference between groups was analyzed using two-tail t test.

Article Snippet: Rabbit polyclonal anti-EZH2 antibody was purchased from Cell Signaling Technology; rabbit polyclonal anti-H3K27me3, mouse mAb to 5mC and rabbit mAb to 5hmC antibodies were purchased from Abcam; mouse mAb to DNMT1 and rat mAb to DNMT3A antibodies were purchased from Active Motif.

Techniques: Expressing, Modification, Immunofluorescence, Fluorescence

Journal: Nature Communications

Article Title: Gestational diabetes mellitus causes genome hyper-methylation of oocyte via increased EZH2

doi: 10.1038/s41467-024-55499-x

Figure Lengend Snippet: a , b inhibitors of EZH2 are used to inhibit the function of EZH2 in oocytes, and the H3K27me3 level is examined using immunofluorescence; control, n = 83; Dznep, n = 73 (p = 0.009185); Gsk343, n = 75 (p = 0.043357); c – e after inhibition of EZH2, the 5mC and 5hmC level in MII oocytes are examined using immunofluorescence; n: control=44, Dznep=56 (p = 0.007128), and Gsk343 = 36 (p = 0.021183) for 5mC, and control=42, Dznep=44 (p = 0.000235), and Gsk343 = 25 (p = 0.000834) for 5hmC; f , g Abca1 and Dact3 are target genes of EZH2, and the DNA methylation level of Abca1 (p = 0.000137 and 0.037005) and Dact3 (p = 0.017237 and 0.04808) is reduced by inhibiting the function of EZH2; data presented as percentage, and the statistical difference was examined using chi-square test; h – j when Ezh2 expression is knocked down in oocytes using siRNA, the genomic methylation level of 5mC and 5hmC is examined; n: control =27 and Ezh2 -siRNA = 58 (p = 0.010829) for 5mC, and control=13 and Ezh2 -siRNA =56 (p = 0.006307) for 5hmC; k – m with Ezh2 overexpression in oocytes, the 5mC and 5hmC level are examined using immunofluorescence; n: control =50 and Ezh2 -overexpressionn=63 for both of 5mC (p = 1.31743*10 −5 ) and 5hmC (p = 0.001759). *p < 0.05; **p < 0.01; ***p < 0.001; black circle, methylated CG; white circle, unmethylated CG. Bar, 20 μm. data presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal anti-EZH2 antibody was purchased from Cell Signaling Technology; rabbit polyclonal anti-H3K27me3, mouse mAb to 5mC and rabbit mAb to 5hmC antibodies were purchased from Abcam; mouse mAb to DNMT1 and rat mAb to DNMT3A antibodies were purchased from Active Motif.

Techniques: Immunofluorescence, Control, Inhibition, DNA Methylation Assay, Expressing, Methylation, Over Expression

Journal: Nature Communications

Article Title: Gestational diabetes mellitus causes genome hyper-methylation of oocyte via increased EZH2

doi: 10.1038/s41467-024-55499-x

Figure Lengend Snippet: a , b EZH2 colocalizes with DNMT1 and DNMT3a in nucleus of GV oocytes; c – e Ezh2 expression knockdown reduces the level of EZH2 and DNMT1 at chromatin; n: control=48 and Ezh2 -siRNA=47 for EZH2 (p = 5.07772*10 −8 ), and control=19 and Ezh2 -siRNA=27 for DNMT1 (p = 2.62159 × 10 −5 ); and f – h Ezh2 overexpression increases the level of EZH2 and DNMT1 at chromatin, n: control=57 and Ezh2 -overexpression=45 for EZH2 (p = 4.54368 × 10 −7 ), and control=26 and Ezh2 -overexpression=22 for DNMT1 (p = 3.05373*10 −7 ); but i – l the level of DNMT3a at chromatin is not affected by Ezh2 knockdown (n: control=25 and Ezh2 -siRNA=20, p = 0.20404) and overexpression (n: control=31 and Ezh2 -overexpression=22, p = 0.227746). data presented as mean ± SEM. ****p < 0.00001; ns, no significant difference. Bar, 20 μm. Ezh2 -ov, Ezh2 overexpression; ns, no significant difference. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal anti-EZH2 antibody was purchased from Cell Signaling Technology; rabbit polyclonal anti-H3K27me3, mouse mAb to 5mC and rabbit mAb to 5hmC antibodies were purchased from Abcam; mouse mAb to DNMT1 and rat mAb to DNMT3A antibodies were purchased from Active Motif.

Techniques: Expressing, Knockdown, Control, Over Expression

Journal: Nature Communications

Article Title: Gestational diabetes mellitus causes genome hyper-methylation of oocyte via increased EZH2

doi: 10.1038/s41467-024-55499-x

Figure Lengend Snippet: a , b the interaction of EZH2 with DNMT1 and DNMT3A and COCs (n = 2000) and oocytes (n = 5300) is examined using co-immunoprecipitation and Western-blotting; c the interaction between EZH2 and DNMT1 and DNMT3A in ovaries (n = 8) is examined using co-immunoprecipitation; d , e EZH2 is knockdown using siRNA in oocytes and the H3K27me3 level is examined using immunofluorescence (n: control=43 and Ezh2 -siRNA=46, p = 0.002896); f , g SUZ12 knockdown in oocytes is performed using siRNA, and the H3K27me3 level is examined (n: control=43 and Ezh2 -siRNA=48, p = 0.034347). *p < 0.05; **p < 0.01. Bar, 20 μm. H3K27me3, histone 3 lysine 27 trimethylation. data presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal anti-EZH2 antibody was purchased from Cell Signaling Technology; rabbit polyclonal anti-H3K27me3, mouse mAb to 5mC and rabbit mAb to 5hmC antibodies were purchased from Abcam; mouse mAb to DNMT1 and rat mAb to DNMT3A antibodies were purchased from Active Motif.

Techniques: Immunoprecipitation, Western Blot, Knockdown, Immunofluorescence, Control

Journal: Nature Communications

Article Title: Gestational diabetes mellitus causes genome hyper-methylation of oocyte via increased EZH2

doi: 10.1038/s41467-024-55499-x

Figure Lengend Snippet: a the dynamics of EZH2 in follicular development of GDF1 are examined using immunofluorescence, and b the relative level of EZH2 in GDF1 is compared with NGDF1; n: NGDF1 = 23 and GDF1 = 20 for primordial follicles (p = 0.088426), NGDF1 = 25 and GDF1 = 22 for primary follicles (p = 0.896508), NGDF1 = 43 and GDF1 = 47 for secondary follicles (p = 0.942229), and NGDF1 = 16 and GDF1 = 15 for antral follicles (p = 0.67194); 8 ovaries from 8 mice were used; c H3K27me3 level in folliculogenesis of GDF1 is examined, and d the relative level of it is compared with NGDF1; n: NGDF1 = 23 and GDF1 = 22 for primordial follicles (p = 0.003264), NGDF1 = 34 and GDF1 = 30 for primary follicles (p = 1.65546*10 −5 ), NGDF1 = 30 and GDF1 = 25 for secondary follicles (p = 0.018598), and NGDF1 = 17 and GDF1 = 15 for antral follicles (p = 0.008278); 8 ovaries from 8 mice were used; e protein levels of DNMT3A and EZH2 in NGDF1 and GDF1 oocytes are examined using Western-blotting; f , g the level of DNMT3a in GDF1 oocytes is not affected by GDM compared with NGDF1 (n: NGDF1 = 19 and GDF1 = 18, p = 0.256458); h , i the mRNA expression of Dnmt1 (p = 0.655248), Uhrf1 (p = 0.898845) and Stella (p = 0.018583) in GDF1 oocytes (data presented as mean ± SD), but j , k the level of DNMT1 at chromatin is increased in GDF1 oocytes (n: NGDF1 = 14 and GDF1 = 15, p = 0.000605); l – n Ezh2 knockdown in GDF1 oocytes significantly decreased the level of 5mC and 5hmC (n: NGDF1 = 20, GDF1 = 30 (p = 0.041405), and Ezh2 -siRNA=16 (p = 0.005935) for 5mC,, and NGDF1 = 23, GDF1 = 30 (p = 0.017439), and Ezh2 -siRNA=16 (p = 0.034593) for 5hmC, p = ). *p < 0.05; **p < 0.01; ***p < 0.001; ns, no significant difference. Bar, 20 μm. data presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal anti-EZH2 antibody was purchased from Cell Signaling Technology; rabbit polyclonal anti-H3K27me3, mouse mAb to 5mC and rabbit mAb to 5hmC antibodies were purchased from Abcam; mouse mAb to DNMT1 and rat mAb to DNMT3A antibodies were purchased from Active Motif.

Techniques: Immunofluorescence, Western Blot, Expressing, Knockdown

Journal: Nature communications

Article Title: EZH2 mutations in follicular lymphoma distort H3K27me3 profiles and alter transcriptional responses to PRC2 inhibition.

doi: 10.1038/s41467-024-47701-x

Figure Lengend Snippet: Fig. 2 | Ezh2Y641F is sufficient to drive aberrant H3K27me3 patterns and strengthened H3K27ac peaks. a, b Western blot analysis in isogenic iMEFs. Identical samples were run in parallel on different blots. Source data are provided as a Source Data file. c In vitro histone methyltransferase assay using 3H-labeled S- adenosyl-methionine comparing the activity of reconstituted PRC2 variants on recombinant nucleosomes in the presence of JARID2, AEBP2 or H3K27me3 peptide. Source data are provided as a Source Data file. d H3K27me3 and H3K27me2 CUT&RUN tracks for WT and Ezh2Y641F/WT iMEFs with corresponding genomic annotations shown below (note that the H3K27me3 signal for WT cells exceeds the range displayed). e Heatmap of changes in enrichment for histone modifications across 10 emission states defined by ChromHMM (see Supplementary Fig. 1j) comparing Ezh2Y641F/WT iMEFs to WT. f H3K27ac CUT&RUN tracks for WT and Ezh2Y641F/WT iMEFs with corresponding genomic annotations shown below. g Violin

Article Snippet: The resulting Table 1 | List of antibodies used in the study Antibody Host Application Source Clone/identifier EZH2 rabbit polyclonal WB homemade33 N/A H3K27me3 rabbit monoclonal WB, C&R,ChIP CST C36B11 H3K27me2 mouse monoclonal WB, C&R Active Motif 324 H3K27me1 mouse monoclonal WB Active Motif 321 EED rabbit polyclonal WB homemade33 N/A H3.3K27M rabbit monoclonal WB Millipore RM192 H2Aub rabbit monoclonal C&R CST 8240S H3K27ac rabbit polyclonal C&R Abcam Ab4729 H3K4me3 rabbit monoclonal C&R CST C42D8 H3K36me3 rabbit polyclonal C&R Abcam Ab9050 H4 rabbit polyclonal WB Active Motif AB_2636967 SUZ12 rabbit monoclonal C&R CST D39F6 Rabbit IgG goat polyclonal WB secondary BioRad 12004161 Mouse IgG goat polyclonal WB secondary BioRad STAR117D800GA WB Western blot, C&R CUT&RUN, ChIP chromatin immunoprecipitation, CST Cell Signaling Technology.

Techniques: Western Blot, In Vitro, HMT Assay, Labeling, Activity Assay, Recombinant