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Active Motif rabbit anti-cdk8
Rabbit Anti Cdk8, supplied by Active Motif, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti-cdk8/pm35715442-399-21-24?v=Active+Motif
Average 90 stars, based on 1 article reviews

rabbit anti-cdk8 - by Bioz Stars, 2026-08

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A. THZ531, but not inhibitors targeting CDK8 (CCT251545), or CDK9 (NVP-2), or BET family of bromodomain proteins (JQ1) increases MYC expression. SKBR3 cells were treated with indicated inhibitors (200 nM) for 4 hours, followed by fluorescent immunoblotting. B. HCC1954 cells were treated with indicated inhibitors (all 200 nM, except 500 nM for flavopiridol) for 4 hours, followed by fluorescent immunoblotting. C. HCC1954 cells were treated with NVP-2 for 4 hours at indicated concentrations. Note that NVP-2 does not obviously induce MYC expression at low doses, and totally abolished MYC expression at 40 or 200 nM. D. HCC1954 cells were treated with increasing concentrations of flavopiridol for 4 hours. Lysates were prepared and subjected to fluorescent immunoblotting. E. HCC1954 were treated with vehicle (0.08% DMSO, v/v), CDK8/19 inhibitor CCT251545 at the indicated concentrations, or THZ531 (400 nM). Four hours post treatment, cells were lysed with 1x SDS sample buffer, and cell lysates were subjected to fluorescent immunoblotting using the indicated antibodies. The molecular weights of the fluorescent protein markers and clone identities for monoclonal antibodies are indicated. Merged images show signals from two primary antibodies raised in different species. Note that for all immunoblotting based on short duration of treatment, cells were lysed with the same amount of sample buffer for complete lysis, and the same volume of lysates was loaded onto each lane of SDS-PAGE gels. Thus, each lane represents signal from similar number of cells.

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. THZ531, but not inhibitors targeting CDK8 (CCT251545), or CDK9 (NVP-2), or BET family of bromodomain proteins (JQ1) increases MYC expression. SKBR3 cells were treated with indicated inhibitors (200 nM) for 4 hours, followed by fluorescent immunoblotting. B. HCC1954 cells were treated with indicated inhibitors (all 200 nM, except 500 nM for flavopiridol) for 4 hours, followed by fluorescent immunoblotting. C. HCC1954 cells were treated with NVP-2 for 4 hours at indicated concentrations. Note that NVP-2 does not obviously induce MYC expression at low doses, and totally abolished MYC expression at 40 or 200 nM. D. HCC1954 cells were treated with increasing concentrations of flavopiridol for 4 hours. Lysates were prepared and subjected to fluorescent immunoblotting. E. HCC1954 were treated with vehicle (0.08% DMSO, v/v), CDK8/19 inhibitor CCT251545 at the indicated concentrations, or THZ531 (400 nM). Four hours post treatment, cells were lysed with 1x SDS sample buffer, and cell lysates were subjected to fluorescent immunoblotting using the indicated antibodies. The molecular weights of the fluorescent protein markers and clone identities for monoclonal antibodies are indicated. Merged images show signals from two primary antibodies raised in different species. Note that for all immunoblotting based on short duration of treatment, cells were lysed with the same amount of sample buffer for complete lysis, and the same volume of lysates was loaded onto each lane of SDS-PAGE gels. Thus, each lane represents signal from similar number of cells.

Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).

Techniques: Expressing, Western Blot, Bioprocessing, Lysis, SDS Page

Transfection efficiency determined by flow cytometry . The transfection efficiency was 97.2% 6 h after transfecting with CDK8-siRNA of HCT116. The ratio of Lipofectin 2000 to siRNA was 4 μL: 4 μL, and the concentration of CDK8-siRNA is 80 pmol/L.

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: Transfection efficiency determined by flow cytometry . The transfection efficiency was 97.2% 6 h after transfecting with CDK8-siRNA of HCT116. The ratio of Lipofectin 2000 to siRNA was 4 μL: 4 μL, and the concentration of CDK8-siRNA is 80 pmol/L.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Transfection, Flow Cytometry, Concentration Assay

CDK8 and β-catenin mRNA expression of CDK-siRNA transfected HCT116 cells detected by RT-PCR . 48 h later of CDK8-siRNA transfection, RT-PCR was performed to detect CDK8 and β-catenin mRNA expression. A: CDK8-siRNA group; B: scrambled siRNA group; C: non-siRNA group; D, E and F represented corresponding internal reference, and M: marker. Results are given as average value of the gray in three target genes and interal controls from three independent experiments.

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: CDK8 and β-catenin mRNA expression of CDK-siRNA transfected HCT116 cells detected by RT-PCR . 48 h later of CDK8-siRNA transfection, RT-PCR was performed to detect CDK8 and β-catenin mRNA expression. A: CDK8-siRNA group; B: scrambled siRNA group; C: non-siRNA group; D, E and F represented corresponding internal reference, and M: marker. Results are given as average value of the gray in three target genes and interal controls from three independent experiments.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Marker

Representative Western blots of CDK8 and β-catenin expression level in CDK-siRNA transfected HCT116 cells . 72 h later of CDK8-siRNA transfection of HCT116 cells, protein expression of CDK8 (A) and β-catenin (B) was determined by western blot assay. a: non-siRNA group; b: scrambled siRNA group; c: CDK-siRNA group. Results are given as average value of the gray in three target genes and interal controls from three independent experiments.

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: Representative Western blots of CDK8 and β-catenin expression level in CDK-siRNA transfected HCT116 cells . 72 h later of CDK8-siRNA transfection of HCT116 cells, protein expression of CDK8 (A) and β-catenin (B) was determined by western blot assay. a: non-siRNA group; b: scrambled siRNA group; c: CDK-siRNA group. Results are given as average value of the gray in three target genes and interal controls from three independent experiments.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Western Blot, Expressing, Transfection

Cell proliferation activity after transfection of CDK8-siRNA assessed by MTT assay . Curves of cell growth after transfection for 24, 48 and 72 h by MTT assay. Results are given as means ± SD from three independent experiments. P < 0.05.

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: Cell proliferation activity after transfection of CDK8-siRNA assessed by MTT assay . Curves of cell growth after transfection for 24, 48 and 72 h by MTT assay. Results are given as means ± SD from three independent experiments. P < 0.05.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Activity Assay, Transfection, MTT Assay

Effect of CDK8-siRNA transfection on the apoptosis and cell cycle of HCT116 cells . 48 h after transfection, cell apoptosis (A) and cell cycle (B) were determined by flow cytometry. Quadrants D2-D4 represent necrotic/late apoptotic cells, viable cells, and early apoptotic cells, respectively. Results are given as means ± SD from three independent experiments.

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: Effect of CDK8-siRNA transfection on the apoptosis and cell cycle of HCT116 cells . 48 h after transfection, cell apoptosis (A) and cell cycle (B) were determined by flow cytometry. Quadrants D2-D4 represent necrotic/late apoptotic cells, viable cells, and early apoptotic cells, respectively. Results are given as means ± SD from three independent experiments.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Transfection, Flow Cytometry

CDK8 and β-catenin mRNA expression in colon tumor and adjacent normal tissues detected by real-time PCR . Fresh tumor and corresponding adjacent tissues from 12 patients were resected under sterile conditions and then snapfrozen in liquid nitrogen immediately. 200 mg tissue was taken out from liquid nitrogen and plused 1 ml Trizol when RNA was extaracted. Real-time PCR is performed for the expression levels of CDK8 (A) and β-catenin (B). Results are given as means ± SD from three independent experiments. P < 0.05.

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: CDK8 and β-catenin mRNA expression in colon tumor and adjacent normal tissues detected by real-time PCR . Fresh tumor and corresponding adjacent tissues from 12 patients were resected under sterile conditions and then snapfrozen in liquid nitrogen immediately. 200 mg tissue was taken out from liquid nitrogen and plused 1 ml Trizol when RNA was extaracted. Real-time PCR is performed for the expression levels of CDK8 (A) and β-catenin (B). Results are given as means ± SD from three independent experiments. P < 0.05.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction

CDK8 and β-catenin protein expression in colon tumor and adjacent normal tissues detected by IHC . The expression of CDK8 (left) and β-catenin (right) was stained brown and present in tumor tissue and adjacent normal tissues. Representative sites with negative (a, 400 X ), moderate positive (c, 400 × ), strongly positive (e, 400 ×) expression of CDK8 and corresponding weakly positive (b, 400 ×), moderate positive (d, 400 ×), strongly positive (f, 400 ×) expression of β-catenin.

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: CDK8 and β-catenin protein expression in colon tumor and adjacent normal tissues detected by IHC . The expression of CDK8 (left) and β-catenin (right) was stained brown and present in tumor tissue and adjacent normal tissues. Representative sites with negative (a, 400 X ), moderate positive (c, 400 × ), strongly positive (e, 400 ×) expression of CDK8 and corresponding weakly positive (b, 400 ×), moderate positive (d, 400 ×), strongly positive (f, 400 ×) expression of β-catenin.

Article Snippet: Rabbit anti-human CDK8 antibody, rabbit anti-human β-catenin antibody, and rat anti-human β-actin antibody were purchased from Chemicon (USA).

Techniques: Expressing, Staining

a, A representative Western blot analysis of CDK8 immunoprecipitation (n = 2) from nuclear extracts from Med13/13l fl/fl (WT) and Med13/13l −/− (CKM-Mediator KO) ESCs, probed with the indicated antibodies. b, Quality control metrics of the Hi-C data, showing total sequenced read-pairs in millions, total valid contacts in millions and percentages in cis contacts for WT and CKM-Mediator KO ESCs. c, Aggregate analysis of super enhancer interactions in WT and CKM-Mediator KO ESCs. The difference between WT and KO is shown. d, Aggregate analysis of Hi-C signal (10 kb resolution) at pairs of Polycomb domains at the indicated distance ranges in Med13/13l fl/fl (WT) and Med13/13l −/− (CKM-Mediator KO) ESCs, with 200 kb flanking regions. Interactions of inactive non-Polycomb gene promoters subsampled to match regions as in Fig. (n = 2096), are included as a negative control (bottom). The difference between WT and KO is shown. e , Capture-C interaction scores for interactions between Polycomb domains in WT and CKM-Mediator KO ESCs (number of promoters = 51, number of interactions = 148). f, Boxplot analysis of Capture-C interaction scores from WT and CKM-Mediator KO ESCs showing interactions between Polycomb gene promoters and other Polycomb-domains (left), or non-Polycomb gene promoters with active sites (H3K27ac, right). Number of promoters (P) and interactions (int) is shown. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians.

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a, A representative Western blot analysis of CDK8 immunoprecipitation (n = 2) from nuclear extracts from Med13/13l fl/fl (WT) and Med13/13l −/− (CKM-Mediator KO) ESCs, probed with the indicated antibodies. b, Quality control metrics of the Hi-C data, showing total sequenced read-pairs in millions, total valid contacts in millions and percentages in cis contacts for WT and CKM-Mediator KO ESCs. c, Aggregate analysis of super enhancer interactions in WT and CKM-Mediator KO ESCs. The difference between WT and KO is shown. d, Aggregate analysis of Hi-C signal (10 kb resolution) at pairs of Polycomb domains at the indicated distance ranges in Med13/13l fl/fl (WT) and Med13/13l −/− (CKM-Mediator KO) ESCs, with 200 kb flanking regions. Interactions of inactive non-Polycomb gene promoters subsampled to match regions as in Fig. (n = 2096), are included as a negative control (bottom). The difference between WT and KO is shown. e , Capture-C interaction scores for interactions between Polycomb domains in WT and CKM-Mediator KO ESCs (number of promoters = 51, number of interactions = 148). f, Boxplot analysis of Capture-C interaction scores from WT and CKM-Mediator KO ESCs showing interactions between Polycomb gene promoters and other Polycomb-domains (left), or non-Polycomb gene promoters with active sites (H3K27ac, right). Number of promoters (P) and interactions (int) is shown. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians.

Article Snippet: Antibodies used for Western blot analysis were rabbit polyclonal anti-MED13L (A302-420A, Bethyl laboratories), rabbit polyclonal anti-MED13 (GTX129674, Genetex), rabbit monoclonal anti-CDK8 (ab229192, Abcam), rabbit polyclonal anti-CCNC (A301-989A, Bethyl laboratories), rabbit polyclonal anti-MED1 (A300-793A, Bethyl laboratories), rabbit polyclonal anti-MED15 (A302-422A, Bethyl laboratories), rabbit polyclonal anti-MED23 (A300-425A, Bethyl laboratories), rabbit polyclonal anti-MED17 (GTX115241, Genetex), rabbit polyclonal anti-MED14 (A301-044A-T, Bethyl laboratories), rabbit monoclonal anti-RING1B (5694, Cell Signaling), rabbit monoclonal anti-SUZ12 (3737, Cell Signaling), rabbit polyclonal anti-PCGF2 (sc-10744, Santa Cruz), rabbit monoclonal anti-T7-Tag (D9E1X, 13246, Cell Signaling), mouse monoclonal anti-TBP (ab818, Abcam), rabbit monoclonal anti-HDAC1 (ab109411, Abcam), and mouse monoclonal anti-Flag (F1804, Sigma).

Techniques: Western Blot, Immunoprecipitation, Hi-C, Negative Control, Capture-C

a , A Venn diagram showing the overlap between CDK8 peaks and Polycomb domains. Number of peaks and percent overlap are indicated. b, Metaplot analysis of CDK8 enrichment at Polycomb domains (n = 2097) in WT and CKM-Mediator (CKM-MED) KO ESCs. c , Heatmaps showing CDK8 ChIPseq signal at Polycomb domains (n = 2097) in WT and CKM-Mediator KO ESCs, sorted by decreasing RING1B signal. d, A representative Western blot analysis (n = 6) of nuclear extracts from WT and CKM-MED KO ESCs probed with the indicated antibodies. TBP and HDAC1 are used as loading controls. e , Comparison between loss of Hi-C signal (difference between CKM-MED-KO and WT) and loss of cPRC1 (PCGF2) binding (log2 fold change) at Polycomb domains. Polycomb domains were divided into equal bins (261 domains each) based on log2 fold change in cPRC1 binding. f, Comparison between loss of Hi-C signal (difference between CKM-MED-KO and WT) and levels of CDK8 binding in WT cells (log2RPKM). Domains were divided into eight bins based on CDK8 RPKM levels.

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a , A Venn diagram showing the overlap between CDK8 peaks and Polycomb domains. Number of peaks and percent overlap are indicated. b, Metaplot analysis of CDK8 enrichment at Polycomb domains (n = 2097) in WT and CKM-Mediator (CKM-MED) KO ESCs. c , Heatmaps showing CDK8 ChIPseq signal at Polycomb domains (n = 2097) in WT and CKM-Mediator KO ESCs, sorted by decreasing RING1B signal. d, A representative Western blot analysis (n = 6) of nuclear extracts from WT and CKM-MED KO ESCs probed with the indicated antibodies. TBP and HDAC1 are used as loading controls. e , Comparison between loss of Hi-C signal (difference between CKM-MED-KO and WT) and loss of cPRC1 (PCGF2) binding (log2 fold change) at Polycomb domains. Polycomb domains were divided into equal bins (261 domains each) based on log2 fold change in cPRC1 binding. f, Comparison between loss of Hi-C signal (difference between CKM-MED-KO and WT) and levels of CDK8 binding in WT cells (log2RPKM). Domains were divided into eight bins based on CDK8 RPKM levels.

Techniques: Western Blot, Hi-C, Binding Assay

a , Heatmaps showing RING1B (PRC1) and CDK8 ChIP–seq signals at Polycomb domains ( n = 2097), sorted by decreasing RING1B signal. b , A genomic snapshot of a Polycomb-bound locus, showing CDK8, RING1B, PCGF2, CBX7 and H3K27me3 ChIP–seq signal in WT (+) and CKM–MED KO (-) ESCs. c , Heatmaps showing RING1B, PCGF2, CBX7 and H3K27me3 ChIP–seq signal at Polycomb domains ( n = 2,097) in WT (+) and CKM–MED KO (-) ESCs, sorted by decreasing RING1B signal. d , Metaplot analysis of RING1B, PCGF2, CBX7 and H3K27me3 enrichment at Polycomb domains ( n = 2,097) in WT and CKM–MED KO ESCs.

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a , Heatmaps showing RING1B (PRC1) and CDK8 ChIP–seq signals at Polycomb domains ( n = 2097), sorted by decreasing RING1B signal. b , A genomic snapshot of a Polycomb-bound locus, showing CDK8, RING1B, PCGF2, CBX7 and H3K27me3 ChIP–seq signal in WT (+) and CKM–MED KO (-) ESCs. c , Heatmaps showing RING1B, PCGF2, CBX7 and H3K27me3 ChIP–seq signal at Polycomb domains ( n = 2,097) in WT (+) and CKM–MED KO (-) ESCs, sorted by decreasing RING1B signal. d , Metaplot analysis of RING1B, PCGF2, CBX7 and H3K27me3 enrichment at Polycomb domains ( n = 2,097) in WT and CKM–MED KO ESCs.

Techniques: ChIP-sequencing

a , A schematic of the integrated TetO site and experimental setup. b , A snapshot showing Capture-C read count signal from TetR-PCGF2, TetR-CDK8 and TetR-GFP lines at the TetO array. CDK8 and PCGF2 (cPRC1) ChIP–seq signal is given as a reference. The TetO bait is shown as a triangle and interactions created with surrounding cPRC1-bound sites are represented with arrowheads. c , A schematic of the cPRC1 ( Pcgf4 −/− Pcgf2 fl/fl ) conditional KO line. d , A snapshot showing Capture-C read count signal from WT and cPRC1 KO ESCs. Interactions between the Nkx2-1 promoter bait (triangle) and surrounding Polycomb domain sites are shown with arrowheads. cPRC1 binding (PCGF2 ChIP–seq) is shown as a reference. e , Boxplot analysis of normalized read counts from WT and cPRC1 KO ESCs showing interactions between Polycomb gene promoters and other Polycomb domains (left), or non-Polycomb gene promoters and active sites (H3K27ac, right). Boxes show IQR, center lines represent the median, whiskers extend 1.5 × IQR or the most extreme point (whichever is closer to the median), whereas notches extend by 1.58 x IQR/sqrt( n ), giving a roughly 95% confidence interval for comparing medians.

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a , A schematic of the integrated TetO site and experimental setup. b , A snapshot showing Capture-C read count signal from TetR-PCGF2, TetR-CDK8 and TetR-GFP lines at the TetO array. CDK8 and PCGF2 (cPRC1) ChIP–seq signal is given as a reference. The TetO bait is shown as a triangle and interactions created with surrounding cPRC1-bound sites are represented with arrowheads. c , A schematic of the cPRC1 ( Pcgf4 −/− Pcgf2 fl/fl ) conditional KO line. d , A snapshot showing Capture-C read count signal from WT and cPRC1 KO ESCs. Interactions between the Nkx2-1 promoter bait (triangle) and surrounding Polycomb domain sites are shown with arrowheads. cPRC1 binding (PCGF2 ChIP–seq) is shown as a reference. e , Boxplot analysis of normalized read counts from WT and cPRC1 KO ESCs showing interactions between Polycomb gene promoters and other Polycomb domains (left), or non-Polycomb gene promoters and active sites (H3K27ac, right). Boxes show IQR, center lines represent the median, whiskers extend 1.5 × IQR or the most extreme point (whichever is closer to the median), whereas notches extend by 1.58 x IQR/sqrt( n ), giving a roughly 95% confidence interval for comparing medians.

Techniques: Capture-C, ChIP-sequencing, Binding Assay

a , A representative Western blot analysis (n = 3) of nuclear extracts from the TetR-fusion lines used for Capture-C analysis probed with anti-Flag antibody to detect expression of the fusion proteins. HDAC1 is used as a loading control. b , ChIP-qPCR analysis of binding of the different TetR-fusion lines to the TetO array. Data are presented as mean value (n = 2) ±SD. Data points for individual replicates are shown. c, ChIP-qPCR analysis of binding of the CKM-Mediator complex to the TetO array in the TetR-CDK8, TetR-PCGF2, and TetR-GFP lines.. Data are presented as mean value (n = 2 for TetR-CDK8 and n = 3 for TetR-PCGF2 and TetR-GFP) ± SD. Data points for individual replicates are shown. d , Boxplot analysis of Capture-C mean normalised read counts and interaction scores in the TetR-fusion lines, looking at interactions with Polycomb domains (PCGF2-bound). Number of interactions is shown. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians. e , Snapshots showing Capture-C read count signal from TetR-CDK8, TetR-PCGF2 and TetR-GFP lines at a control locus. CDK8 and PCGF2 (cPRC1) ChIPseq signal is given as a reference. The Fli1 promoter bait is shown as a triangle and interactions created with surrounding cPRC1-bound sites are represented with arrowheads. f , A representative Western blot analysis of nuclear extracts (n = 3) from WT and cPRC1 KO ESCs probed with the indicated antibodies. TBP is used as a loading control. g , Metaplot analysis of CDK8 enrichment at CDK8 peaks (n = 24275) and Polycomb domains (n = 2097) in WT and cPRC1 KO ESCs. h , Heatmaps showing CDK8 ChIPseq signal at CDK8 peaks (n = 24275) and Polycomb domains (n = 2097) in WT and cPRC1 KO ESCs, sorted by decreasing CDK8 or RING1B signal, respectively. i, Boxplot analysis of Capture-C interaction scores from WT and cPRC1 KO ESCs showing interactions between Polycomb gene promoters and other Polycomb-domains (left), or non-Polycomb gene promoters and active sites (H3K27ac, right). Number of promoters (P) and interactions (int) is shown. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians.

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a , A representative Western blot analysis (n = 3) of nuclear extracts from the TetR-fusion lines used for Capture-C analysis probed with anti-Flag antibody to detect expression of the fusion proteins. HDAC1 is used as a loading control. b , ChIP-qPCR analysis of binding of the different TetR-fusion lines to the TetO array. Data are presented as mean value (n = 2) ±SD. Data points for individual replicates are shown. c, ChIP-qPCR analysis of binding of the CKM-Mediator complex to the TetO array in the TetR-CDK8, TetR-PCGF2, and TetR-GFP lines.. Data are presented as mean value (n = 2 for TetR-CDK8 and n = 3 for TetR-PCGF2 and TetR-GFP) ± SD. Data points for individual replicates are shown. d , Boxplot analysis of Capture-C mean normalised read counts and interaction scores in the TetR-fusion lines, looking at interactions with Polycomb domains (PCGF2-bound). Number of interactions is shown. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians. e , Snapshots showing Capture-C read count signal from TetR-CDK8, TetR-PCGF2 and TetR-GFP lines at a control locus. CDK8 and PCGF2 (cPRC1) ChIPseq signal is given as a reference. The Fli1 promoter bait is shown as a triangle and interactions created with surrounding cPRC1-bound sites are represented with arrowheads. f , A representative Western blot analysis of nuclear extracts (n = 3) from WT and cPRC1 KO ESCs probed with the indicated antibodies. TBP is used as a loading control. g , Metaplot analysis of CDK8 enrichment at CDK8 peaks (n = 24275) and Polycomb domains (n = 2097) in WT and cPRC1 KO ESCs. h , Heatmaps showing CDK8 ChIPseq signal at CDK8 peaks (n = 24275) and Polycomb domains (n = 2097) in WT and cPRC1 KO ESCs, sorted by decreasing CDK8 or RING1B signal, respectively. i, Boxplot analysis of Capture-C interaction scores from WT and cPRC1 KO ESCs showing interactions between Polycomb gene promoters and other Polycomb-domains (left), or non-Polycomb gene promoters and active sites (H3K27ac, right). Number of promoters (P) and interactions (int) is shown. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians.

Techniques: Western Blot, Capture-C, Expressing, Binding Assay

a , A schematic of the differentiation of WT and CKM–MED KO ESCs used for cnRNA-seq. b , Boxplot analysis of the expression of CKM–MED-dependent genes ( n = 631) in WT ESCs and following RA (retinoic acid) induction (WT and CKM–MED KO). Boxes show IQR, center lines represent the median, whiskers extend by 1.5 × IQR or the most extreme point (whichever is closer to the median), whereas notches extend by 1.58 x IQR/sqrt( n ), giving a roughly 95% confidence interval for comparing medians. c , A schematic of the differentiation of WT and cPRC1 KO ESCs for cnRNA-seq. d , As in b but for cPRC1 cKO cells. e , A screenshot showing the expression of genes within the HoxB cluster following RA induction of CKM–MED cKO or cPRC1 KO cells. Forward strand is shown on top and reverse strand is shown at the bottom of each track. ChIP–seq tracks for CDK8 and cPRC1 (PCGF2) enrichment are shown. f , Boxplot analysis of the expression of RA-induced (RA-ind) genes from the Polycomb (PcG) network (top, n=482) and CKM–Med-dependent genes from the PcG network (bottom, n=184) following RA induction of CKM–MED cKO or cPRC1 KO cells. Boxes are defined as in a .

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a , A schematic of the differentiation of WT and CKM–MED KO ESCs used for cnRNA-seq. b , Boxplot analysis of the expression of CKM–MED-dependent genes ( n = 631) in WT ESCs and following RA (retinoic acid) induction (WT and CKM–MED KO). Boxes show IQR, center lines represent the median, whiskers extend by 1.5 × IQR or the most extreme point (whichever is closer to the median), whereas notches extend by 1.58 x IQR/sqrt( n ), giving a roughly 95% confidence interval for comparing medians. c , A schematic of the differentiation of WT and cPRC1 KO ESCs for cnRNA-seq. d , As in b but for cPRC1 cKO cells. e , A screenshot showing the expression of genes within the HoxB cluster following RA induction of CKM–MED cKO or cPRC1 KO cells. Forward strand is shown on top and reverse strand is shown at the bottom of each track. ChIP–seq tracks for CDK8 and cPRC1 (PCGF2) enrichment are shown. f , Boxplot analysis of the expression of RA-induced (RA-ind) genes from the Polycomb (PcG) network (top, n=482) and CKM–Med-dependent genes from the PcG network (bottom, n=184) following RA induction of CKM–MED cKO or cPRC1 KO cells. Boxes are defined as in a .

Techniques: Expressing, ChIP-sequencing

a, A schematic illustration of the generation of the T7-MED14 expressing Med13/13l fl/fl ESC line. b, PCR showing amplification of homozygously-tagged T7-Med14 alleles (n = 2). c, A representative Western blot analysis of nuclear extracts from the T7-MED14 Med13/13l fl/fl ESC line, following tamoxifen (TAM) treatment (n = 3). Extract from an untagged ESC line was used as a control. HDAC1 was used as a loading control. d, A representative immunoprecipitation (IP) of endogenously T7-tagged MED14 with T7 antibody using nuclear extracts from Med13/13l fl/f ESCs before (UNT) and after tamoxifen (TAM) treatment (n = 2). The IPs were probed with the indicated antibodies. An IP from an untagged ESC line was performed as a negative control and a Western blot for SUZ12 was included as a control protein that does not interact with Mediator. e, Heatmaps of CDK8 and T7-MED14 ChIPseq signal at Polycomb domains (n = 2097) and H3K27ac peaks (n = 4037), sorted by decreasing CDK8 signal. f, Boxplots showing gene expression change (log2FC) of CKM-Mediator-dependent (n = 631) and CKM-Mediator-independent (n = 2689) RA-induced genes following RA differentiation of WT ESCs. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians. g, Boxplots showing T7-MED14 ChIPseq signal at the TSS (1000 bp) of the different classes of RA-induced gene classes as defined in e in ESCs and RA-induced cells (WT and CKM-Mediator KO). Boxes are defined as in f . Signal is an average from three independent biological experiments.

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a, A schematic illustration of the generation of the T7-MED14 expressing Med13/13l fl/fl ESC line. b, PCR showing amplification of homozygously-tagged T7-Med14 alleles (n = 2). c, A representative Western blot analysis of nuclear extracts from the T7-MED14 Med13/13l fl/fl ESC line, following tamoxifen (TAM) treatment (n = 3). Extract from an untagged ESC line was used as a control. HDAC1 was used as a loading control. d, A representative immunoprecipitation (IP) of endogenously T7-tagged MED14 with T7 antibody using nuclear extracts from Med13/13l fl/f ESCs before (UNT) and after tamoxifen (TAM) treatment (n = 2). The IPs were probed with the indicated antibodies. An IP from an untagged ESC line was performed as a negative control and a Western blot for SUZ12 was included as a control protein that does not interact with Mediator. e, Heatmaps of CDK8 and T7-MED14 ChIPseq signal at Polycomb domains (n = 2097) and H3K27ac peaks (n = 4037), sorted by decreasing CDK8 signal. f, Boxplots showing gene expression change (log2FC) of CKM-Mediator-dependent (n = 631) and CKM-Mediator-independent (n = 2689) RA-induced genes following RA differentiation of WT ESCs. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians. g, Boxplots showing T7-MED14 ChIPseq signal at the TSS (1000 bp) of the different classes of RA-induced gene classes as defined in e in ESCs and RA-induced cells (WT and CKM-Mediator KO). Boxes are defined as in f . Signal is an average from three independent biological experiments.

Techniques: Expressing, Amplification, Western Blot, Immunoprecipitation, Negative Control

a , A genomic snapshot of two CKM–Mediator-dependent genes, showing CDK8 and T7-MED14 ChIP–seq and cnRNA-seq in WT (+) and CKM–MED KO (-) ESCs (top) and following RA induction (bottom). b , Heatmaps showing CDK8 and T7-MED14 ChIP–seq signal at promoters (TSS±2.5 kb) of CKM–MED-dependent genes in ESCs and following RA induction ( n = 631). T7-MED14 signals are shown for WT and CKM–Mediator KO RA-induced cells. Genes are sorted by decreasing T7-MED14 signal in RA-treated cells. Metaplots showing read density are shown on the top of each heatmap.

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a , A genomic snapshot of two CKM–Mediator-dependent genes, showing CDK8 and T7-MED14 ChIP–seq and cnRNA-seq in WT (+) and CKM–MED KO (-) ESCs (top) and following RA induction (bottom). b , Heatmaps showing CDK8 and T7-MED14 ChIP–seq signal at promoters (TSS±2.5 kb) of CKM–MED-dependent genes in ESCs and following RA induction ( n = 631). T7-MED14 signals are shown for WT and CKM–Mediator KO RA-induced cells. Genes are sorted by decreasing T7-MED14 signal in RA-treated cells. Metaplots showing read density are shown on the top of each heatmap.

Techniques: ChIP-sequencing

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