dmap1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc dmap1
    Dmap1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dmap1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc dmap1
    Dmap1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dmap1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc dmap1
    Bub3 interacts with <t>DMAP1</t> during mitotic arrest. In A-F, immunoblotting analyses were performed using the indicated antibodies; Data represent 1 out of 3 experiments. a , HPDE cells expressing Flag-Bub3 were synchronized in interphase by thymidine double block (2 mM) or were synchronized in mitosis by nocodazole (200 nM) treatment for 16 h after releasing thymidine double block for 8 h (left panel), then cells were extracted and subjected to immunoprecipitation with an anti-Flag antibody. The precipitates from immunoprecipitated-Flag-bub3 were washed by Flag peptides and then were analyzed by coomassie brilliant blue staining and immunoblotting (right panel). b , HPDE cells were synchronized by thymidine double block (2 mM) and were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h, cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. c , HPDE cells were transfected with or without DMAP1 siRNA. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. d , HPDE and PANC-1 cells were synchronized by thymidine double block (2 mM) and were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h (left panel). PANC-1 cells were treated with SU6656 (shown as ‘SU’) (10 μM) for 1 h post Nocodazole treatment for 16 h. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. e , HPDE cells overexpressed with Src Y527F were synchronized by thymidine double block (2 mM) and were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h (left panel). Cellular extracts subjected to immunoprecipitation with an anti-Bub3 antibody. f , HPDE cells synchronized by thymidine double block (2 mM) were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h (left panel). Cells expressing HA-DMAP1was transfected with plasmids expressing indicated length of Flag-Bub3. Cellular extracts were subjected to immunoprecipitation with an anti-Flag antibody
    Dmap1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "C-Src confers resistance to mitotic stress through inhibition DMAP1/Bub3 complex formation in pancreatic cancer"

    Article Title: C-Src confers resistance to mitotic stress through inhibition DMAP1/Bub3 complex formation in pancreatic cancer

    Journal: Molecular Cancer

    doi: 10.1186/s12943-018-0919-5

    Bub3 interacts with DMAP1 during mitotic arrest. In A-F, immunoblotting analyses were performed using the indicated antibodies; Data represent 1 out of 3 experiments. a , HPDE cells expressing Flag-Bub3 were synchronized in interphase by thymidine double block (2 mM) or were synchronized in mitosis by nocodazole (200 nM) treatment for 16 h after releasing thymidine double block for 8 h (left panel), then cells were extracted and subjected to immunoprecipitation with an anti-Flag antibody. The precipitates from immunoprecipitated-Flag-bub3 were washed by Flag peptides and then were analyzed by coomassie brilliant blue staining and immunoblotting (right panel). b , HPDE cells were synchronized by thymidine double block (2 mM) and were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h, cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. c , HPDE cells were transfected with or without DMAP1 siRNA. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. d , HPDE and PANC-1 cells were synchronized by thymidine double block (2 mM) and were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h (left panel). PANC-1 cells were treated with SU6656 (shown as ‘SU’) (10 μM) for 1 h post Nocodazole treatment for 16 h. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. e , HPDE cells overexpressed with Src Y527F were synchronized by thymidine double block (2 mM) and were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h (left panel). Cellular extracts subjected to immunoprecipitation with an anti-Bub3 antibody. f , HPDE cells synchronized by thymidine double block (2 mM) were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h (left panel). Cells expressing HA-DMAP1was transfected with plasmids expressing indicated length of Flag-Bub3. Cellular extracts were subjected to immunoprecipitation with an anti-Flag antibody
    Figure Legend Snippet: Bub3 interacts with DMAP1 during mitotic arrest. In A-F, immunoblotting analyses were performed using the indicated antibodies; Data represent 1 out of 3 experiments. a , HPDE cells expressing Flag-Bub3 were synchronized in interphase by thymidine double block (2 mM) or were synchronized in mitosis by nocodazole (200 nM) treatment for 16 h after releasing thymidine double block for 8 h (left panel), then cells were extracted and subjected to immunoprecipitation with an anti-Flag antibody. The precipitates from immunoprecipitated-Flag-bub3 were washed by Flag peptides and then were analyzed by coomassie brilliant blue staining and immunoblotting (right panel). b , HPDE cells were synchronized by thymidine double block (2 mM) and were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h, cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. c , HPDE cells were transfected with or without DMAP1 siRNA. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. d , HPDE and PANC-1 cells were synchronized by thymidine double block (2 mM) and were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h (left panel). PANC-1 cells were treated with SU6656 (shown as ‘SU’) (10 μM) for 1 h post Nocodazole treatment for 16 h. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. e , HPDE cells overexpressed with Src Y527F were synchronized by thymidine double block (2 mM) and were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h (left panel). Cellular extracts subjected to immunoprecipitation with an anti-Bub3 antibody. f , HPDE cells synchronized by thymidine double block (2 mM) were released for 8 h, followed by nocodazole (200 nM) treatment for 16 h (left panel). Cells expressing HA-DMAP1was transfected with plasmids expressing indicated length of Flag-Bub3. Cellular extracts were subjected to immunoprecipitation with an anti-Flag antibody

    Techniques Used: Western Blot, Expressing, Blocking Assay, Immunoprecipitation, Staining, Transfection

    c-Src phosphorylates DMAP1 at Tyr246 and disrupts Bub3/DMAP1 complex formation. In a - e , immunoblotting analyses were performed using the indicated antibodies. Data represent 1 out of 3 experiments. In a , c and d , cells were synchronized in interphase (I) by thymidine double block (2 mM) or were synchronized in mitosis (M) by nocodazole (200 nM) treatment for 16 h after releasing thymidine double block for 8 h. In a - e , data represent 1 out of 3 experiments. a , PANC-1 or SW1990 cells were synchronized in interphase or mitosis. Cellular extracts were subjected to immunoprecipitation with an anti-Src antibody. b , In vitro phosphorylation analyses were performed by mixing the purified active c-Src with the indicated purified GST-DMAP1 proteins in the presence of [γ- 32 P]ATP. Tyr246 of DMAP1 is evolutionarily conserved in the indicated species ( b , left panel). c , Cells synchronized in mitosis were expressed with indicated Flag-DMAP1. Cellular extracts were subjected to immunoprecipitation with an anti-Flag antibody Cells. d , The indicated purified GST-DMAP1 protein was mixed with mitotic extracts from PANC-1 cells in the presence or absence of c-Src. GST pull down analyses were performed. e , Purified His-Bub3 protein with or without phosphorylation by c-Src was mixed with GST-DMAP1 purified protein with or without p38. GST pull down analyses were performed
    Figure Legend Snippet: c-Src phosphorylates DMAP1 at Tyr246 and disrupts Bub3/DMAP1 complex formation. In a - e , immunoblotting analyses were performed using the indicated antibodies. Data represent 1 out of 3 experiments. In a , c and d , cells were synchronized in interphase (I) by thymidine double block (2 mM) or were synchronized in mitosis (M) by nocodazole (200 nM) treatment for 16 h after releasing thymidine double block for 8 h. In a - e , data represent 1 out of 3 experiments. a , PANC-1 or SW1990 cells were synchronized in interphase or mitosis. Cellular extracts were subjected to immunoprecipitation with an anti-Src antibody. b , In vitro phosphorylation analyses were performed by mixing the purified active c-Src with the indicated purified GST-DMAP1 proteins in the presence of [γ- 32 P]ATP. Tyr246 of DMAP1 is evolutionarily conserved in the indicated species ( b , left panel). c , Cells synchronized in mitosis were expressed with indicated Flag-DMAP1. Cellular extracts were subjected to immunoprecipitation with an anti-Flag antibody Cells. d , The indicated purified GST-DMAP1 protein was mixed with mitotic extracts from PANC-1 cells in the presence or absence of c-Src. GST pull down analyses were performed. e , Purified His-Bub3 protein with or without phosphorylation by c-Src was mixed with GST-DMAP1 purified protein with or without p38. GST pull down analyses were performed

    Techniques Used: Western Blot, Blocking Assay, Immunoprecipitation, In Vitro, Purification

    p38 phosphorylates Bub3 at Ser211 and promotes Bub3/DMAP1interaction. In a - h , immunoblotting analyses were performed using the indicated antibodies. Data represent 1 out of 3 experiments. In a - d and h , cells were synchronized in interphase (I) by thymidine (2 mM) double block or were synchronized in mitosis (M) by nocodazole (200 nM) treatment for 16 h after releasing thymidine double block for 8 h. In a - h , data represent 1 out of 3 experiments. a , HPDE cells were synchronized in interphase or mitosis. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody and the immunoprecipitates were treated with or without CIP (10 units). b , HPDE cells synchronized in interphase or mitosis were treated with Compound C (10 μM), SP600125 (20 μM) and SB203580 (25 μM) for 1 h, after Nocodazole treatment for 16 h. c , PANC-1 or SW1990 cells were synchronized in interphase or mitosis. Cells were treated with SB203580 (25 μM) and SU6656 (shown as ‘SU’) (10 μM) for 1 h, after Nocodazole treatment for 16 h. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. d , HPDE or PANC-1 cells were synchronized in interphase or mitosis. Cellular extracts were subjected to immunoprecipitation with an anti-p38 antibody. e and f , In vitro phosphorylation analyses were performed by mixing the purified active p38 with the indicated purified GST-Bub3 proteins in the presence of [γ-32P]ATP. Ser211 of Bub3 is evolutionarily conserved in the indicated species ( f , left panel). g , The indicated purified His-Bub3 protein was mixed with GST-DMAP1 purified protein with or without p38. GST pull down analyses were performed. h , HPDE cells synchronized in mitosis were expressed with indicated Flag-Bub3. Cellular extracts were subjected to immunoprecipitation with an anti-Flag antibody
    Figure Legend Snippet: p38 phosphorylates Bub3 at Ser211 and promotes Bub3/DMAP1interaction. In a - h , immunoblotting analyses were performed using the indicated antibodies. Data represent 1 out of 3 experiments. In a - d and h , cells were synchronized in interphase (I) by thymidine (2 mM) double block or were synchronized in mitosis (M) by nocodazole (200 nM) treatment for 16 h after releasing thymidine double block for 8 h. In a - h , data represent 1 out of 3 experiments. a , HPDE cells were synchronized in interphase or mitosis. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody and the immunoprecipitates were treated with or without CIP (10 units). b , HPDE cells synchronized in interphase or mitosis were treated with Compound C (10 μM), SP600125 (20 μM) and SB203580 (25 μM) for 1 h, after Nocodazole treatment for 16 h. c , PANC-1 or SW1990 cells were synchronized in interphase or mitosis. Cells were treated with SB203580 (25 μM) and SU6656 (shown as ‘SU’) (10 μM) for 1 h, after Nocodazole treatment for 16 h. Cellular extracts were subjected to immunoprecipitation with an anti-Bub3 antibody. d , HPDE or PANC-1 cells were synchronized in interphase or mitosis. Cellular extracts were subjected to immunoprecipitation with an anti-p38 antibody. e and f , In vitro phosphorylation analyses were performed by mixing the purified active p38 with the indicated purified GST-Bub3 proteins in the presence of [γ-32P]ATP. Ser211 of Bub3 is evolutionarily conserved in the indicated species ( f , left panel). g , The indicated purified His-Bub3 protein was mixed with GST-DMAP1 purified protein with or without p38. GST pull down analyses were performed. h , HPDE cells synchronized in mitosis were expressed with indicated Flag-Bub3. Cellular extracts were subjected to immunoprecipitation with an anti-Flag antibody

    Techniques Used: Western Blot, Blocking Assay, Immunoprecipitation, In Vitro, Purification

    Bub3/DMAP1 complex represses anti-apoptotic genes transcription. In a , immunoblotting analyses were performed using the indicated antibodies; data represent 1 out of 3 experiments. In c - e , the values represent mean ± s.e.m. of three independent experiments. a , SW1990 cells were double blocked by thymide and treated with nocodazole (200 nM) following by releasing for the indicated periods. b , SW1990 cells were released for 4 h after thymidine double block and nocodazole (200 nM) for 16 h. Hierachical clustering of 4307 probe sets correlating with DMAP1 Y246F-expressed cells show that genes relevant to anti-apoptosis or autophagy were effective in separating cases from DMAP1 WT-expressed cells. c and d SW1990 cells expressed with the indicated plasmids were treated with nocodazole (200 nM) post thymidine double block, and were released for the indicated time. Relative mRNA levels were analyzed by real-time PCR. In c , * represents p < 0.05 between groups of cells expressing rDMAP1 Y246F plus WT rBub3 and groups of cells expressing rDMAP1 Y246F plus rBub3 S211A. In d , * represents p < 0.05 between groups of cells expressing rDMAP1 Y246F and groups of cells expressing rDMAP1 Y246F plus DNMT1 siRNA. e , Cell apoptosis was analyzed by Annexin V assays followed by flow cytometry. ** represents p < 0.01 between indicated groups
    Figure Legend Snippet: Bub3/DMAP1 complex represses anti-apoptotic genes transcription. In a , immunoblotting analyses were performed using the indicated antibodies; data represent 1 out of 3 experiments. In c - e , the values represent mean ± s.e.m. of three independent experiments. a , SW1990 cells were double blocked by thymide and treated with nocodazole (200 nM) following by releasing for the indicated periods. b , SW1990 cells were released for 4 h after thymidine double block and nocodazole (200 nM) for 16 h. Hierachical clustering of 4307 probe sets correlating with DMAP1 Y246F-expressed cells show that genes relevant to anti-apoptosis or autophagy were effective in separating cases from DMAP1 WT-expressed cells. c and d SW1990 cells expressed with the indicated plasmids were treated with nocodazole (200 nM) post thymidine double block, and were released for the indicated time. Relative mRNA levels were analyzed by real-time PCR. In c , * represents p < 0.05 between groups of cells expressing rDMAP1 Y246F plus WT rBub3 and groups of cells expressing rDMAP1 Y246F plus rBub3 S211A. In d , * represents p < 0.05 between groups of cells expressing rDMAP1 Y246F and groups of cells expressing rDMAP1 Y246F plus DNMT1 siRNA. e , Cell apoptosis was analyzed by Annexin V assays followed by flow cytometry. ** represents p < 0.01 between indicated groups

    Techniques Used: Western Blot, Blocking Assay, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry

    DMAP1 Y246 phosphorylation is required for tumourigenesis. a and b , A total of 5 × 10 6 SW1990 cells with Bub3 or DMAP1 depletion and reconstituted expression of the WT rBub3/WT rDMAP1, WT rBub3/rDMAP1 Y246F or rBub3 S211A/rDMAP1 Y246F were subcutaneously injected into the athymic nude mice. Paclitaxel (5 mg/kg) was injected intraperitoneally every two days once the volume of tumours reached 200 mm 3 . Data represent the mean ± s.e.m. ( n = 8). *represents p < 0.05 and **represents p < 0.01 between indicated groups. Representative tumour xenografts were shown ( a ). Tumour volumes and weight were measured. In the measurement of tumour volumes, length (a) and width (b) and calculated using the following equation: V = ab 2 /2. Data represent the means ± s.e.m. ( n = 8, right panel) ( b ). c and d Immunoblotting analyses ( c ) and Immunohistochemical staining ( d ) with anti-DMAP1 pY246 was performed on human pancreas tumour specimens. Representative photos were shown. For immunoblotting analyses, the numbers underneath the blotting bands represent the normalized density quantified by using densitometry using Image J 2 × software. e , The survival times for 90 patients with low (0–2 staining scores, blue curve) versus high (2.1–5 staining scores, red curve) DMAP1 Tyr246 phosphorylation (low, 29 patients; high, 61 patients) (upper right) were compared. The Kaplan-Meier method and log-rank tests indicating the significance level of the association of DMAP1 Tyr246 phosphorylation ( p = 0.014) with patient survival. The table (lower) shows the cox-multivariate analysis after adjustment for patient sex and age, indicating the significance level of the association of DMAP1 Tyr246 phosphorylation ( p = 0.0247, HR = 1.942) with patient survival. f , The schematic diagram showing the regulatory role of Bub3/DMAP1 complex in DNA methylation upon mitotic stress in normal pancreatic epithelial cells and pancreatic cancer cells. Mitotic arrest induces p38 activation, which phosphorylates Bub3 at Ser 211 and promotes its interaction with DMAP1/DNMT1. TAp73 recruits Bub3/DMAP1 complex to promoter regions of its-targeted genes, where DMAP1/DNMT1 mediates DNA methylation and blocks expression of genes responsible for anti-apoptosis. c-Src can phosphorylate DMAP1 at Tyr246, which disrupts Bub3/DMAP1 complex formation. In normal cells with limited c-Src activity, p38/Bub3/DMAP1 signaling is readily activated under mitotic arrest; in turn, cells are prone to apoptosis (left part). In cancer cells, hyperactive c-Src leads to DMAP1 tyr246 phosphorylation and inhibits mitotic arrest-induced cell apoptosis mediated by p38/Bub3/DMAP1 signaling (right part)
    Figure Legend Snippet: DMAP1 Y246 phosphorylation is required for tumourigenesis. a and b , A total of 5 × 10 6 SW1990 cells with Bub3 or DMAP1 depletion and reconstituted expression of the WT rBub3/WT rDMAP1, WT rBub3/rDMAP1 Y246F or rBub3 S211A/rDMAP1 Y246F were subcutaneously injected into the athymic nude mice. Paclitaxel (5 mg/kg) was injected intraperitoneally every two days once the volume of tumours reached 200 mm 3 . Data represent the mean ± s.e.m. ( n = 8). *represents p < 0.05 and **represents p < 0.01 between indicated groups. Representative tumour xenografts were shown ( a ). Tumour volumes and weight were measured. In the measurement of tumour volumes, length (a) and width (b) and calculated using the following equation: V = ab 2 /2. Data represent the means ± s.e.m. ( n = 8, right panel) ( b ). c and d Immunoblotting analyses ( c ) and Immunohistochemical staining ( d ) with anti-DMAP1 pY246 was performed on human pancreas tumour specimens. Representative photos were shown. For immunoblotting analyses, the numbers underneath the blotting bands represent the normalized density quantified by using densitometry using Image J 2 × software. e , The survival times for 90 patients with low (0–2 staining scores, blue curve) versus high (2.1–5 staining scores, red curve) DMAP1 Tyr246 phosphorylation (low, 29 patients; high, 61 patients) (upper right) were compared. The Kaplan-Meier method and log-rank tests indicating the significance level of the association of DMAP1 Tyr246 phosphorylation ( p = 0.014) with patient survival. The table (lower) shows the cox-multivariate analysis after adjustment for patient sex and age, indicating the significance level of the association of DMAP1 Tyr246 phosphorylation ( p = 0.0247, HR = 1.942) with patient survival. f , The schematic diagram showing the regulatory role of Bub3/DMAP1 complex in DNA methylation upon mitotic stress in normal pancreatic epithelial cells and pancreatic cancer cells. Mitotic arrest induces p38 activation, which phosphorylates Bub3 at Ser 211 and promotes its interaction with DMAP1/DNMT1. TAp73 recruits Bub3/DMAP1 complex to promoter regions of its-targeted genes, where DMAP1/DNMT1 mediates DNA methylation and blocks expression of genes responsible for anti-apoptosis. c-Src can phosphorylate DMAP1 at Tyr246, which disrupts Bub3/DMAP1 complex formation. In normal cells with limited c-Src activity, p38/Bub3/DMAP1 signaling is readily activated under mitotic arrest; in turn, cells are prone to apoptosis (left part). In cancer cells, hyperactive c-Src leads to DMAP1 tyr246 phosphorylation and inhibits mitotic arrest-induced cell apoptosis mediated by p38/Bub3/DMAP1 signaling (right part)

    Techniques Used: Expressing, Injection, Western Blot, Immunohistochemical staining, Staining, Software, DNA Methylation Assay, Activation Assay, Activity Assay

    c-Src-mediated DMAP1 phosphorylation blocks DMAP1-mediated DNA methylation. a , SW1990 cells expressed with the indicated plasmids were synchronized in mitosis (M) by nocodazole (200 nM) treatment for 16 h after releasing thymidine double block for 8 h. DNA methylation levels of promoters and CpG islands or CpG islands shores were presented as ratio of methylated reads to unmethylated reads. The values represent from 2 repeated samples. b , SW1990 cells expressed with the indicated plasmids were synchronized in mitosis (M) by nocodazole (200 nM) treatment. DNA methylation profile of the promoter region (TSS ±1 kb) of BCL2L1 . The values represent from 2 repeated samples. c , SW1990 and PANC-1 cells expressed with the indicated plasmids were synchronized in mitosis (M) by nocodazole (200 nM) treatment. 5-mc levels were examined by bisulfite sequencing using primers covering TAp73 binding site of BCL2L1 gene promoter region. d - g , Cells were synchronized in interphase (I) by thymidine (2 mM) double block or were synchronized in mitosis (M) by nocodazole (200 nM) treatment. d , SW1990 cells with depleted DMAP1, and reconstituted expression of WT rDMAP1 or rDMAP1 Y246F were transfected with plasmid for expression of TAp73 shRNA. Relative mRNA level was analyzed by real-time PCR. e , ChIP analyses were performed in SW1990 cells. The primers covering TAp73 binding site of BCL2L1 gene promoter region were used for the real-time PCR. f , SW1990 cells were transfected with plasmid for expression of TAp73 shRNA. ChIP analyses were performed. The primers covering TAp73 binding site of BCL2L1 gene promoter region were used for the real-time PCR. g , SW1990 cells were expressed with the indicated plasmids. ChIP analyses were performed. The primers covering TAp73 binding site of BCL2L1 gene promoter region were used for the real-time PCR. The y axis shows the value normalized to the input. The values represent mean ± s.e.m. of three independent experiments;*represents p < 0.05 and **represents p < 0.01 between indicated groups
    Figure Legend Snippet: c-Src-mediated DMAP1 phosphorylation blocks DMAP1-mediated DNA methylation. a , SW1990 cells expressed with the indicated plasmids were synchronized in mitosis (M) by nocodazole (200 nM) treatment for 16 h after releasing thymidine double block for 8 h. DNA methylation levels of promoters and CpG islands or CpG islands shores were presented as ratio of methylated reads to unmethylated reads. The values represent from 2 repeated samples. b , SW1990 cells expressed with the indicated plasmids were synchronized in mitosis (M) by nocodazole (200 nM) treatment. DNA methylation profile of the promoter region (TSS ±1 kb) of BCL2L1 . The values represent from 2 repeated samples. c , SW1990 and PANC-1 cells expressed with the indicated plasmids were synchronized in mitosis (M) by nocodazole (200 nM) treatment. 5-mc levels were examined by bisulfite sequencing using primers covering TAp73 binding site of BCL2L1 gene promoter region. d - g , Cells were synchronized in interphase (I) by thymidine (2 mM) double block or were synchronized in mitosis (M) by nocodazole (200 nM) treatment. d , SW1990 cells with depleted DMAP1, and reconstituted expression of WT rDMAP1 or rDMAP1 Y246F were transfected with plasmid for expression of TAp73 shRNA. Relative mRNA level was analyzed by real-time PCR. e , ChIP analyses were performed in SW1990 cells. The primers covering TAp73 binding site of BCL2L1 gene promoter region were used for the real-time PCR. f , SW1990 cells were transfected with plasmid for expression of TAp73 shRNA. ChIP analyses were performed. The primers covering TAp73 binding site of BCL2L1 gene promoter region were used for the real-time PCR. g , SW1990 cells were expressed with the indicated plasmids. ChIP analyses were performed. The primers covering TAp73 binding site of BCL2L1 gene promoter region were used for the real-time PCR. The y axis shows the value normalized to the input. The values represent mean ± s.e.m. of three independent experiments;*represents p < 0.05 and **represents p < 0.01 between indicated groups

    Techniques Used: DNA Methylation Assay, Blocking Assay, Methylation, Methylation Sequencing, Binding Assay, Expressing, Transfection, Plasmid Preparation, shRNA, Real-time Polymerase Chain Reaction

    vimentin  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc vimentin
    Vimentin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti dmap1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti dmap1
    Anti Dmap1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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