cbp  (Santa Cruz Biotechnology)

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers.

doi: 10.18632/oncotarget.2430

Figure Lengend Snippet: Figure 2: The effect of CBP on hTERT promoter activity, hTERT protein expression and telomerase activity. (A) Lung cancer cells were co-transfected with the plasmids of hTERT promoter driven-luciferase and pCDNA3.1-CBP or pCDNA3.1-Lac Z for 48 h followed by a dual-luciferase assay. The relative luciferase intensity per mg protein was calculated in the treated cells. (B) Lung cancer cells were co-transfected with hTERT promoter driven-luciferase and CBP siRNA or control siRNA for 48 h followed by a dual-luciferase assay. The relative luciferase intensity per mg protein was calculated in the treated cells. (C) Up-regulation of hTERT protein expression by the overexpression of CBP. H1299 cells were treated with pcDNA3.1-CBP for 48 h, and the expression of CBP itself in the nucleus and hTERT protein in the cytoplasm were analyzed by Western blot in lung cancer cells. (D, E) Downregulation of hTERT protein expression by the knockdown of CBP expression. H1299 cells were transfected with a CBP-specific siRNA for 48 h, the expression of CBP itself in the nucleus and hTERT protein in the cytoplasm were analyzed by Western blot (D), and the telomerase activity was assessed (E). All of the measurements represent the means ± SE of three independent experiments. *, P < 0.05, significant differences between treatment groups and DMSO control groups.

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

Techniques: Activity Assay, Expressing, Transfection, Luciferase, Control, Over Expression, Western Blot, Knockdown

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers.

doi: 10.18632/oncotarget.2430

Figure Lengend Snippet: Figure 3: The effect of CBP on lung tumor growth in vitro and in vivo. (A) H1299 cells were transfected with CBP overexpression vector pcDNA3.1-CBP. At different time points after transfection, cell viability was measured by MTT assay. (B) H1299 cells were treated with CBP specific siRNA or inhibitor. At different time points after treatment, cell viability was measured by MTT assay. The mean and SE obtained from three independent experiments are plotted (*, P < 0.05,**, P < 0.01). (C) The nude mice containing xenografts of human lung cancer were intratumorally treated with non-specific control siRNA or CBP-specific siRNA, and the tumor volumes ± SE were calculated at different days after treatment. (N=5; *, P < 0.05). (D) Immunohistochemistry of CBP and hTERT from tumor xenografts in nonspecific control siRNA- and CBP-specific siRNA-treated nude mice (400x magnification).

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

Techniques: In Vitro, In Vivo, Transfection, Over Expression, Plasmid Preparation, MTT Assay, Control, Immunohistochemistry

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers.

doi: 10.18632/oncotarget.2430

Figure Lengend Snippet: Figure 6: The interaction of CBP with Sp1 and AP-2 and the acetylationt of Sp1 by CBP in lung cancer cells. (A) The nuclear extracts of human lung normal and cancer cells were prepared for immunoprecipitation using an antibody against Sp1 or AP-2β and then evaluated by immunoblot using antibody against CBP. (B) Human lung cancer H1299 cells grown on chamber slides were cultivated for 24 h, and the subcellular localization and the colocalization of CBP with Sp1 or AP-2β were examined by confocal microscopy analysis with a confocal microscope. More than 100 cells were inspected per experiment, and cells with typical morphology were presented. (C) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done. Sp1 was tested in the pulled down proteins by immunoblot using antibody against Sp1. (D) Chromatin immunoprecipitation assays were done using antibody against Sp1. PCR products of hTERT promoter (-378 to +60) were separated on 1% agarose gels. The last lane represents the IgG control. (E) Immunoprecipitation was performed using antibody against Sp1. The acetylated Sp1 was determined by immunoblot using the antibody against acetylation. (F) Immunoprecipitation was performed in human lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or inhibitor using antibody against Sp1. The acetylated Sp1 was tested by immunoblot using antibody against acetylation. (G) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done in lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or CBP-specific inhibitor. The level of Sp1 in the pulled down proteins was determined by immunoblot. Densitometric analysis was used to analyze quantitatively the binding activity and acetylation level of Sp1 proteins.

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

Techniques: Immunoprecipitation, Western Blot, Confocal Microscopy, Microscopy, Chromatin Immunoprecipitation, Control, Binding Assay, Activity Assay

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers.

doi: 10.18632/oncotarget.2430

Figure Lengend Snippet: Figure 2: The effect of CBP on hTERT promoter activity, hTERT protein expression and telomerase activity. (A) Lung cancer cells were co-transfected with the plasmids of hTERT promoter driven-luciferase and pCDNA3.1-CBP or pCDNA3.1-Lac Z for 48 h followed by a dual-luciferase assay. The relative luciferase intensity per mg protein was calculated in the treated cells. (B) Lung cancer cells were co-transfected with hTERT promoter driven-luciferase and CBP siRNA or control siRNA for 48 h followed by a dual-luciferase assay. The relative luciferase intensity per mg protein was calculated in the treated cells. (C) Up-regulation of hTERT protein expression by the overexpression of CBP. H1299 cells were treated with pcDNA3.1-CBP for 48 h, and the expression of CBP itself in the nucleus and hTERT protein in the cytoplasm were analyzed by Western blot in lung cancer cells. (D, E) Downregulation of hTERT protein expression by the knockdown of CBP expression. H1299 cells were transfected with a CBP-specific siRNA for 48 h, the expression of CBP itself in the nucleus and hTERT protein in the cytoplasm were analyzed by Western blot (D), and the telomerase activity was assessed (E). All of the measurements represent the means ± SE of three independent experiments. *, P < 0.05, significant differences between treatment groups and DMSO control groups.

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

Techniques: Activity Assay, Expressing, Transfection, Luciferase, Control, Over Expression, Western Blot, Knockdown

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers.

doi: 10.18632/oncotarget.2430

Figure Lengend Snippet: Figure 3: The effect of CBP on lung tumor growth in vitro and in vivo. (A) H1299 cells were transfected with CBP overexpression vector pcDNA3.1-CBP. At different time points after transfection, cell viability was measured by MTT assay. (B) H1299 cells were treated with CBP specific siRNA or inhibitor. At different time points after treatment, cell viability was measured by MTT assay. The mean and SE obtained from three independent experiments are plotted (*, P < 0.05,**, P < 0.01). (C) The nude mice containing xenografts of human lung cancer were intratumorally treated with non-specific control siRNA or CBP-specific siRNA, and the tumor volumes ± SE were calculated at different days after treatment. (N=5; *, P < 0.05). (D) Immunohistochemistry of CBP and hTERT from tumor xenografts in nonspecific control siRNA- and CBP-specific siRNA-treated nude mice (400x magnification).

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

Techniques: In Vitro, In Vivo, Transfection, Over Expression, Plasmid Preparation, MTT Assay, Control, Immunohistochemistry

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers.

doi: 10.18632/oncotarget.2430

Figure Lengend Snippet: Figure 6: The interaction of CBP with Sp1 and AP-2 and the acetylationt of Sp1 by CBP in lung cancer cells. (A) The nuclear extracts of human lung normal and cancer cells were prepared for immunoprecipitation using an antibody against Sp1 or AP-2β and then evaluated by immunoblot using antibody against CBP. (B) Human lung cancer H1299 cells grown on chamber slides were cultivated for 24 h, and the subcellular localization and the colocalization of CBP with Sp1 or AP-2β were examined by confocal microscopy analysis with a confocal microscope. More than 100 cells were inspected per experiment, and cells with typical morphology were presented. (C) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done. Sp1 was tested in the pulled down proteins by immunoblot using antibody against Sp1. (D) Chromatin immunoprecipitation assays were done using antibody against Sp1. PCR products of hTERT promoter (-378 to +60) were separated on 1% agarose gels. The last lane represents the IgG control. (E) Immunoprecipitation was performed using antibody against Sp1. The acetylated Sp1 was determined by immunoblot using the antibody against acetylation. (F) Immunoprecipitation was performed in human lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or inhibitor using antibody against Sp1. The acetylated Sp1 was tested by immunoblot using antibody against acetylation. (G) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done in lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or CBP-specific inhibitor. The level of Sp1 in the pulled down proteins was determined by immunoblot. Densitometric analysis was used to analyze quantitatively the binding activity and acetylation level of Sp1 proteins.

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

Techniques: Immunoprecipitation, Western Blot, Confocal Microscopy, Microscopy, Chromatin Immunoprecipitation, Control, Binding Assay, Activity Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Histone Deacetylase Inhibitors (HDACi) Cause the Selective Depletion of Bromodomain Containing Proteins (BCPs) *

doi: 10.1074/mcp.M114.042499

Figure Lengend Snippet: Depletion of BCPs by RNA i is sufficient to mimic the effects of HDACi treatment onto selected transcripts ( A ), ( B ), ( C ), and ( D ) Bar charts showing transcript abundances of selected target genes (p21, MSL1, GEMI4, and FOXA1) after siRNA-mediated depletion of four BCPs (PBRM1, BRD1, ZMYND11, CREBBP). Transcript levels were quantified by qPCR, and they are expressed as fold changes relatively to a scrambled siRNA control (set to one). For comparison, the fold changes induced by TSA and NaB after 12 h of treatment and quantified by microarray are displayed as dark gray bars. In all the cases shown, the individual depletion of two distinct BPCs induces a change in transcript level that is consistent and of similar effect size to the one induced by HDACi. All the reported values are averages of three biological replicates and error bars indicate the standard error of the mean. Asterisks indicate significant cases (Welch two sample t test p value < .05).

Article Snippet: Cells were allowed to attach to the dish for 24 h then transfected with PBRM1-specific siRNA (#s30400), BRD1-specific siRNA (#s24389), ZMYND11-specific siRNA (#s21153), CREBBP-specific siRNA (#s3495), and negative control no. 1 siRNA (#4390843) purchased from ambion by life technologies.

Techniques: Microarray