anti-5-methylcytosine (5-mc) antibody  (Advisains)

Journal: The Journal of Clinical Investigation

Article Title: The CTBP2-PCIF1 complex regulates m 6 Am modification of mRNA in head and neck squamous cell carcinoma

doi: 10.1172/JCI170173

Figure Lengend Snippet: ( A ) Real-time qPCR analysis of TET2 mRNA expression in control and PCIF1-KO cells ( n = 3). P > 0.05 by 1-way ANOVA with Tukey’s multiple-comparison test. ( B and C ) Real-time qPCR analysis of TET2 mRNA levels at the indicated times in control and PCIF1-KO SCC9 ( B ) and SCC25 ( C ) cells after actinomycin D treatment ( n = 3). P > 0.05 by 2-tailed unpaired Student’s t test. ( D and E ) TET2 expression ( D ) and DNA 5mC and 5hmC modification levels ( E ) were detected in control and PCIF1-KO cells. ( F and G ) TET2 expression ( F ) and DNA 5mC and 5hmC modification levels ( G ) were detected in SCC1 control cells and cells transfected with WT (OE) or mutant PCIF1 plasmid (OE mut ). MB, Methylene blue. ( H ) Schematic diagram of TET2 5′-UTR m 6 Am site mutations. Red arrows denote A594G and A604G mutation. ( I ) Luciferase activity of TET2 5′-UTR WT or TET2 5′-UTR m 6 Am site mutation (5′-UTR MUT) in SCC1 control cells and cells transfected with WT (OE) or mutant PCIF1 plasmid (OE mut ) ( n = 3). P > 0.05, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test. ( J and K ) RNA immunoprecipitation (RIP)–qPCR analysis of TET2 mRNA retrieved by anti-PCIF1 ( J ) and anti-CTBP2 ( K ) antibody in control and PCIF1-KO cells ( n = 3). P > 0.05, *** P < 0.001 by 2-tailed unpaired Student’s t test. ( L ) RIP-qPCR analysis of TET2 mRNA retrieved by anti-PCIF1 antibody in CTBP2-KO cells transfected with vector (sgCTBP2), PCIF1 binding–defective mutant of CTBP2 (ΔCB-1), and WT CTBP2 ( n = 3). P > 0.05, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: After blocking with 5% nonfat milk, the membrane was incubated with anti-5mC (Abcam, ab73938) and anti-5hmC (Proteintech, 40900) antibody at 4°C overnight, followed by secondary antibody.

Techniques: Expressing, Comparison, Modification, Transfection, Mutagenesis, Plasmid Preparation, Luciferase, Activity Assay, Immunoprecipitation, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: The CTBP2-PCIF1 complex regulates m 6 Am modification of mRNA in head and neck squamous cell carcinoma

doi: 10.1172/JCI170173

Figure Lengend Snippet: ( A ) Experimental design of the carcinogen-induced HNSCC mouse model. Sac, sacrifice. ( B ) Diagram of Cre-dependent conditional knockout strategy for Pcif1. ( C ) Representative image of visible tongue lesions in the indicated groups. Scale bar: 1 mm. ( D ) Quantification of HNSCC lesion area in the indicated groups ( n = 8). * P < 0.05, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test. ( E ) Representative H&E staining of HNSCC in the indicated groups. Scale bar: 100 μm. ( F ) Quantification of HNSCC number of lesions in the indicated groups ( n = 8). * P < 0.05, ** P < 0.01, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test. ( G ) Quantification of HNSCC tumor grade in the indicated groups. * P < 0.05, ** P < 0.01 by Pearson’s χ 2 test. ( H ) Representative PCK staining of metastatic lymph node in the indicated groups. Scale bar: 300 μm. ( I ) Quantification of metastatic lymph node percentage in the indicated groups. * P < 0.05 by Pearson’s χ 2 test. ( J – L ) Representative dot blot image ( J ) and quantitative analysis of DNA 5mC ( K ) and 5hmC ( L ) modification levels in the indicated groups ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: After blocking with 5% nonfat milk, the membrane was incubated with anti-5mC (Abcam, ab73938) and anti-5hmC (Proteintech, 40900) antibody at 4°C overnight, followed by secondary antibody.

Techniques: Knock-Out, Comparison, Staining, Dot Blot, Modification

Journal: The Journal of Clinical Investigation

Article Title: The CTBP2-PCIF1 complex regulates m 6 Am modification of mRNA in head and neck squamous cell carcinoma

doi: 10.1172/JCI170173

Figure Lengend Snippet: ( A ) Experimental design of the carcinogen-induced HNSCC mouse model. ( B ) Diagram of Cre-dependent conditional knockout strategy for Ctbp2. ( C ) Representative image of visible tongue lesions in the indicated groups. Scale bar: 1 mm. ( D ) Quantification of HNSCC lesion area in the indicated groups ( n = 8). * P < 0.05, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test. ( E ) Representative H&E staining of HNSCC in the indicated groups. Scale bar: 100 μm. ( F ) Quantification of HNSCC number of lesions in the indicated groups ( n = 8). * P < 0.05, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test. ( G ) Quantification of HNSCC tumor grade in the indicated groups. * P < 0.05 by Pearson’s χ 2 test. ( H ) Representative PCK staining of metastatic lymph node in the indicated groups. Scale bar: 300 μm. ( I ) Quantification of metastatic lymph node percentage in the indicated groups. * P < 0.05 by Pearson’s χ 2 test. ( J – L ) Representative dot blot image ( J ) and quantitative analysis of DNA 5mC ( K ) and 5hmC ( L ) modification levels in the indicated groups ( n = 3). ** P < 0.01, *** P < 0.001 by 1-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: After blocking with 5% nonfat milk, the membrane was incubated with anti-5mC (Abcam, ab73938) and anti-5hmC (Proteintech, 40900) antibody at 4°C overnight, followed by secondary antibody.

Techniques: Knock-Out, Comparison, Staining, Dot Blot, Modification