hpv16/hpv18 e6 antibody (c1p5) - bsa free  (Bio-Techne corporation)

Journal: Oncology Reports

Article Title: Crosstalk between histone modification and DNA methylation orchestrates the epigenetic regulation of the costimulatory factors, Tim-3 and galectin-9, in cervical cancer

doi: 10.3892/or.2019.7388

Figure Lengend Snippet: Primer sequences.

Article Snippet: The antibodies used were as follows: Rabbit polyclonal antibody against human Tim-3 (1:500 dilution, ab185703; Abcam), rabbit polyclonal antibody against human galectin-9 (1:100 dilution, ab123712; Abcam), mouse monoclonal antibody against human DNMT3A (1:200 dilution, sc-365769; Santa Cruz Biotechnology), rabbit monoclonal antibody against human EZH2 (1:1,000 dilution, 5246; Cell Signaling Technology), rabbit monoclonal antibody against human H3K27me3 (1:1,000 dilution, 9733; Cell Signaling Technology), mouse monoclonal antibody against HPV18 E6 (1:250 dilution, NB100-2729; Novus Biologicals) and mouse monoclonal antibody against HPV18 E7 (1:250 dilution, NB110-17215; Novus Biologicals), β-actin mouse monoclonal antibody (1:500 dilution, 60008-1-lg; Proteintech).

Techniques: Sequencing, Control

Journal: Oncology Reports

Article Title: Crosstalk between histone modification and DNA methylation orchestrates the epigenetic regulation of the costimulatory factors, Tim-3 and galectin-9, in cervical cancer

doi: 10.3892/or.2019.7388

Figure Lengend Snippet: Function of HPV18 oncoproteins E6/E7 in the regulation of Tim-3/galectin-9 and DNMT3A expression in human cervical cancer cells. (A) and (B) mRNA levels in of HPV18 E6/E7 -overexpressing C33A cells and of HPV18 E6/E7 -knockdown HeLa cells. (C) and (D) C33A cells were transfected with 18E6/E7/E6/7 lentiviral vector, and HeLa cells were transfected with 18E6/E7/E6/7-specific siRNAs. Western blot analysis was then used to detect DNMT3A expression. (E) Co-IP assay was used to analyzed the incorporation between DNMT3A and HPV18 E6/E7. (F and G) The methylation level of the HAVCR2/LGALS9 promoters was monitored by MS-PCR. Gray level analysis of HAVCR2/LGALS9 methylation levels in cervical cancer cells (M, methylated; U, unmethylated); the methylated and unmethylated levels were quantified as M/M+U% and U/M+U%, respectively. (H and I) C33A cells were transfected with 18E6/E7/E6/7 lentiviral vector, and HeLa cells were transfected with 18E6/E7/E6/7-specific siRNAs. The expression of Tim-3/galectin-9 was determined by western blot analysis. **P<0.01.

Article Snippet: The antibodies used were as follows: Rabbit polyclonal antibody against human Tim-3 (1:500 dilution, ab185703; Abcam), rabbit polyclonal antibody against human galectin-9 (1:100 dilution, ab123712; Abcam), mouse monoclonal antibody against human DNMT3A (1:200 dilution, sc-365769; Santa Cruz Biotechnology), rabbit monoclonal antibody against human EZH2 (1:1,000 dilution, 5246; Cell Signaling Technology), rabbit monoclonal antibody against human H3K27me3 (1:1,000 dilution, 9733; Cell Signaling Technology), mouse monoclonal antibody against HPV18 E6 (1:250 dilution, NB100-2729; Novus Biologicals) and mouse monoclonal antibody against HPV18 E7 (1:250 dilution, NB110-17215; Novus Biologicals), β-actin mouse monoclonal antibody (1:500 dilution, 60008-1-lg; Proteintech).

Techniques: Expressing, Knockdown, Transfection, Plasmid Preparation, Western Blot, Co-Immunoprecipitation Assay, Methylation

Journal: Oncology Reports

Article Title: Crosstalk between histone modification and DNA methylation orchestrates the epigenetic regulation of the costimulatory factors, Tim-3 and galectin-9, in cervical cancer

doi: 10.3892/or.2019.7388

Figure Lengend Snippet: HPV18 oncoprotein E6/E7 alters the expression EZH2 and H3K27me3 in cervical cancer cells. (A and B) C33A cells were transfected with 18E6/E7/E6/7 lentiviral vector and HeLa cells were transfected with 18E6/E7/E6/7 specific siRNAs and western blot analysis was then used to detect EZH2 and H3K27me3 expression. (C) EZH2 and H3K27me3 expression in HeLa and C33A cells detected by western blot analysis. (D) Western blot analysis of the HPV18 E6/E7 protein level in HeLa cells following transfection with siRNAs against EZH2. (E) Co-IP assay was used to analyzed the incorporation between EZH2 and HPV18 E6/E7. (F) Schematic representation of the 4 regions of the EZH2 promoter amplified in the chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) experiment. (G and H) Chromatin was cross-linked, fragmented and immunoprecipitated with either IgG (mock) or anti-E2F-1 and anti-FOXM1 ChIP-grade antibody and the purified DNA was used to amplify with respective primer pairs for indicated four regions in the EZH2 promoters in qPCR. The enrichment of E2F-1 and FOXM1 on EZH2 promoter relative to IgG in HeLa cells, H3 against RPL30 used as positive control. (I and J) Chromatin was cross-linked, fragmented and immunoprecipitated with either IgG (mock) or anti-E2F-1 and anti-FOXM1 ChIP-grade antibody and the purified DNA was used to amplify with respective primer pairs for the indicated 4 regions in the DNMT3A promoters in qPCR. The enrichment of E2F-1 and FOXM1 on DNMT3A promoter relative to IgG in HeLa cells, H3 against RPL30 used as a positive control. **P<0.01; ns, not significant.

Article Snippet: The antibodies used were as follows: Rabbit polyclonal antibody against human Tim-3 (1:500 dilution, ab185703; Abcam), rabbit polyclonal antibody against human galectin-9 (1:100 dilution, ab123712; Abcam), mouse monoclonal antibody against human DNMT3A (1:200 dilution, sc-365769; Santa Cruz Biotechnology), rabbit monoclonal antibody against human EZH2 (1:1,000 dilution, 5246; Cell Signaling Technology), rabbit monoclonal antibody against human H3K27me3 (1:1,000 dilution, 9733; Cell Signaling Technology), mouse monoclonal antibody against HPV18 E6 (1:250 dilution, NB100-2729; Novus Biologicals) and mouse monoclonal antibody against HPV18 E7 (1:250 dilution, NB110-17215; Novus Biologicals), β-actin mouse monoclonal antibody (1:500 dilution, 60008-1-lg; Proteintech).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Co-Immunoprecipitation Assay, Amplification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Purification, Positive Control

Journal: Oncology Reports

Article Title: Crosstalk between histone modification and DNA methylation orchestrates the epigenetic regulation of the costimulatory factors, Tim-3 and galectin-9, in cervical cancer

doi: 10.3892/or.2019.7388

Figure Lengend Snippet: The HPV18 pathway regulates Tim-3/galectin-9 through EZH2-H2K27me3-DNMT3A. HPV18 E6 and E7 regulates EZH2 through direct binding of FOXM1 and E2F-1 the EZH2 promoter in order to activate EZH2 expression; in turn, H3K27me3 is upregulated. EZH2 and H3K27me3 directly interact with the DNMT3A promoter to downregulate its expression. When DNMT3A is decreased, the HAVCR2/LGALS9 promoter region is also demethylated and Tim-3/galectin-9 expression is upregulated.

Article Snippet: The antibodies used were as follows: Rabbit polyclonal antibody against human Tim-3 (1:500 dilution, ab185703; Abcam), rabbit polyclonal antibody against human galectin-9 (1:100 dilution, ab123712; Abcam), mouse monoclonal antibody against human DNMT3A (1:200 dilution, sc-365769; Santa Cruz Biotechnology), rabbit monoclonal antibody against human EZH2 (1:1,000 dilution, 5246; Cell Signaling Technology), rabbit monoclonal antibody against human H3K27me3 (1:1,000 dilution, 9733; Cell Signaling Technology), mouse monoclonal antibody against HPV18 E6 (1:250 dilution, NB100-2729; Novus Biologicals) and mouse monoclonal antibody against HPV18 E7 (1:250 dilution, NB110-17215; Novus Biologicals), β-actin mouse monoclonal antibody (1:500 dilution, 60008-1-lg; Proteintech).

Techniques: Binding Assay, Expressing