Journal: Scientific reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme.

doi: 10.1038/srep39100

Figure Lengend Snippet: Figure 1. Salient characteristics of C>U RNA editing by A3G in 293T cells. (a) Mean and range of editing level at the 712 sites identified as targets for A3G-mediated editing are shown for the three A3G transfectant samples. The sites are ordered by the mean editing level. (b) Logo indicating sequence conservation and base frequency for sequences bearing the editing sites (at position 0). (c) Histogram of nucleotide lengths (b) of inverted repeat sequences flanking the editing sites.

Article Snippet: 2 μ M of 5ʹ Alexa Fluor 488 fluorescent dye-labelled ssDNA substrate of 40 bases (5ʹ -ATTATTATTATTATTATTATTCCCAGGATTTATTTATTTA-3ʹ ) (Integrated DNA Technologies) was incubated at 37 °C for an hour with 100 nM or 1 μ M r A3G (Origene) that was either pre-treated or untreated with Ribonuclease A (Sigma Aldrich) and 2 units of E. coli uracil DNA glycosylase (New England Biolabs) in 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT and 1 mM EDTA in a volume of 10 μ l.1 μ l of 1 N NaOH was added to the reaction, which was then incubated at 37 °C for 15 min. After adding 1 μ l of 1N HCl and 12 μ l of 2X loading buffer (80% formamide, 10X TBE), the samples were incubated at 65 °C for 5 min. 10 μ l of the reaction was electrophoresed on a 10% Mini-PROTEAN TBE-Urea gels (Bio-Rad).

Techniques: Transfection, Sequencing

Journal: Scientific reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme.

doi: 10.1038/srep39100

Figure Lengend Snippet: Figure 2. Transient overexpression of A3G induces C>U RNA editing in 293T transfectants. Sanger chromatograms of cDNAs (in duplicate) and genomic DNAs (gDNA) (in triplicate) from control and A3G transfectants. Edited C is shaded black.

Article Snippet: 2 μ M of 5ʹ Alexa Fluor 488 fluorescent dye-labelled ssDNA substrate of 40 bases (5ʹ -ATTATTATTATTATTATTATTCCCAGGATTTATTTATTTA-3ʹ ) (Integrated DNA Technologies) was incubated at 37 °C for an hour with 100 nM or 1 μ M r A3G (Origene) that was either pre-treated or untreated with Ribonuclease A (Sigma Aldrich) and 2 units of E. coli uracil DNA glycosylase (New England Biolabs) in 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT and 1 mM EDTA in a volume of 10 μ l.1 μ l of 1 N NaOH was added to the reaction, which was then incubated at 37 °C for 15 min. After adding 1 μ l of 1N HCl and 12 μ l of 2X loading buffer (80% formamide, 10X TBE), the samples were incubated at 65 °C for 5 min. 10 μ l of the reaction was electrophoresed on a 10% Mini-PROTEAN TBE-Urea gels (Bio-Rad).

Techniques: Over Expression, Control

Journal: Scientific reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme.

doi: 10.1038/srep39100

Figure Lengend Snippet: Figure 3. Site-specific C>U RNA editing in mRNAs co-purified with rA3G. (a) Immunoblot showing WT A3G expressed in whole cell lysate (20 μg) of 293T cells and recombinant (r) WT A3G (500 ng) obtained from 293T cells (purchased from Origene, Rockville, MD). Full-length blot is presented in Supplementary Fig. S4 (b) Cytidine deamination activity of recombinant A3G was examined in an in vitro reaction with a 5ʹ fluorescent dye-labeled ssDNA substrate of 40 nucleotides (left panel). Full-length gel is presented in Supplementary Fig. S4. Sanger chromatograms of cDNAs of MED1 RNA from in vitro RNA editing assay containing rA3G in the presence of 100 nM (+) or 1 μM (++) and ART MED1 RNA (right panel). (c) Sanger chromatograms of cDNAs of A3G substrates (MED1, KIAA1715, SCD, ITFG1, RFX7, GOLGA5, CHMP4B, CLASP1) (left panel) and A3A substrates (VIM, ASCC2, TMEM179B, SDHB) (right panel) from in vitro RNA editing assay containing only rA3G or from in vitro RNA editing assay containing in vitro transcribed SDHB RNA and purified A3A protein (SDHB lower panel). Edited cytidines are highlighted in black.

Article Snippet: 2 μ M of 5ʹ Alexa Fluor 488 fluorescent dye-labelled ssDNA substrate of 40 bases (5ʹ -ATTATTATTATTATTATTATTCCCAGGATTTATTTATTTA-3ʹ ) (Integrated DNA Technologies) was incubated at 37 °C for an hour with 100 nM or 1 μ M r A3G (Origene) that was either pre-treated or untreated with Ribonuclease A (Sigma Aldrich) and 2 units of E. coli uracil DNA glycosylase (New England Biolabs) in 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT and 1 mM EDTA in a volume of 10 μ l.1 μ l of 1 N NaOH was added to the reaction, which was then incubated at 37 °C for 15 min. After adding 1 μ l of 1N HCl and 12 μ l of 2X loading buffer (80% formamide, 10X TBE), the samples were incubated at 65 °C for 5 min. 10 μ l of the reaction was electrophoresed on a 10% Mini-PROTEAN TBE-Urea gels (Bio-Rad).

Techniques: Purification, Western Blot, Recombinant, Activity Assay, In Vitro, Labeling

Journal: Scientific reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme.

doi: 10.1038/srep39100

Figure Lengend Snippet: Figure 4. Site-directed mutagenesis of A3G shows requirement for both N- and C–terminal domain zinc dependent deaminase motif residues for site-specific RNA editing. (a) Chromatograms of cDNAs (single) from ctrl./A3G transfected 293T cells shows the effect of mutations in A3G-NTD (W94A, C97S, W127A) and A3G-CTD (C291S) on C>U RNA editing (edited C is shaded black) in selected genes. (b) Bar graph showing RNA editing level of GOLGA5, KIAA1715 and MED1 in A3G-NTD and -CTD mutant transfectants. RNA editing levels (ratio of edited versus total RNA) are calculated by SequencherTM software. The detection limit for relative height of minor peaks was 4.86% (depicted by a dotted line). Mean and SEM (n = 3) (c) Immunoblot showing A3G protein expression in whole cell lysates of 293T cells when transfected with empty vector (control), WT or various A3G-NTD and -CTD mutants. The D128K and P129A mutants were run on separate gels on the same day. The dashed line separates the two gels. Full-length blot is presented in Supplementary Fig. S4 (d) Bar graph depicting RNA editing level of KIAA1715, PRPSAP2, SCD and TM7SF3 cDNAs in WT or mutant transfectants. The detection limit is depicted by a dotted line. Mean and SEM (n = 3). Statistical analysis was performed by one- way ANOVA followed by multiple comparisons of RNA editing levels between WT and the mutants. Editing level is considered 0%, when the Sequencher software detects no secondary T peak. ****=adjusted P value ≤ 0.0001; NS = Not Significant.

Article Snippet: 2 μ M of 5ʹ Alexa Fluor 488 fluorescent dye-labelled ssDNA substrate of 40 bases (5ʹ -ATTATTATTATTATTATTATTCCCAGGATTTATTTATTTA-3ʹ ) (Integrated DNA Technologies) was incubated at 37 °C for an hour with 100 nM or 1 μ M r A3G (Origene) that was either pre-treated or untreated with Ribonuclease A (Sigma Aldrich) and 2 units of E. coli uracil DNA glycosylase (New England Biolabs) in 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT and 1 mM EDTA in a volume of 10 μ l.1 μ l of 1 N NaOH was added to the reaction, which was then incubated at 37 °C for 15 min. After adding 1 μ l of 1N HCl and 12 μ l of 2X loading buffer (80% formamide, 10X TBE), the samples were incubated at 65 °C for 5 min. 10 μ l of the reaction was electrophoresed on a 10% Mini-PROTEAN TBE-Urea gels (Bio-Rad).

Techniques: Mutagenesis, Transfection, Software, Western Blot, Expressing, Plasmid Preparation, Control

Journal: Oncology Research

Article Title: Discovery of Two Novel Pyrazole Derivatives as Anticancer Agents Targeting Tubulin Polymerization and MAPK Signaling Pathways

doi: 10.32604/or.2026.074945

Figure Lengend Snippet: Gene expression analysis induced by P3C.1 and P3C.2 in three different cell lines. ( A ) Heatmap of differentially expressed genes of MDA-MB-231, ( B ) Jurkat and ( C ) CEM cells treated with P3C.1 and P3C.2, or with solvent control (DMSO). Each column represents the expression values of the different treatments performed for 6 h for each corresponding cell line, and each row denotes data from one transcript. The color legend of each expression values (log2 fold change) is displayed on the right. ( D ) Differentially expressed genes; up-regulated (green bars) and down-regulated (red bars) obtained for each cell line and treatments, considering only transcripts with a log2 fold change of >1.0, and a p -adjusted value of <0.05.

Article Snippet: In addition, the RAMOS (ATCC, CRL-1596), CEM (ATCC, CCL-119), JURKAT (ATCC, TIB-152), NALM-6 (ATCC, CRL-3273), RPMI-8226, MM.1S, MM.1R, U266, Rec-1, JVM-13, A549 (ATCC, CRM-CCL-185), HCC70 (ATCC, CRL-2315), HCC1419 (ATCC, CRL-2326), T47D (ATCC, CRL-2865), KMS-11, and OVCAR-5 cell lines were grown in Roswell Park Memorial Institute-1640 (RPMI-1640) culture media (Cytiva-Hyclone, SH30027FS, UT, USA,) supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques: Gene Expression, Solvent, Control, Expressing

Journal: Oncology Research

Article Title: Discovery of Two Novel Pyrazole Derivatives as Anticancer Agents Targeting Tubulin Polymerization and MAPK Signaling Pathways

doi: 10.32604/or.2026.074945

Figure Lengend Snippet: P3C.1 and P3C.2 differentially expressed genes induced in common. ( A ) MDA-MB-231, ( B ) Jurkat, and CEM common DEGs identified for both compounds. ( C ) P3C.1 and P3C.2 induced 4 up-regulated genes in common (DUSP8, KLF6, KLF7 and BACH2) in MDA-MB-231, Jurkat and CEM cell lines.

Article Snippet: In addition, the RAMOS (ATCC, CRL-1596), CEM (ATCC, CCL-119), JURKAT (ATCC, TIB-152), NALM-6 (ATCC, CRL-3273), RPMI-8226, MM.1S, MM.1R, U266, Rec-1, JVM-13, A549 (ATCC, CRM-CCL-185), HCC70 (ATCC, CRL-2315), HCC1419 (ATCC, CRL-2326), T47D (ATCC, CRL-2865), KMS-11, and OVCAR-5 cell lines were grown in Roswell Park Memorial Institute-1640 (RPMI-1640) culture media (Cytiva-Hyclone, SH30027FS, UT, USA,) supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques:

Journal: Oncology Research

Article Title: Discovery of Two Novel Pyrazole Derivatives as Anticancer Agents Targeting Tubulin Polymerization and MAPK Signaling Pathways

doi: 10.32604/or.2026.074945

Figure Lengend Snippet: Ingenuity pathways analysis of genes commonly found in P3C.1 and P3C.2-treated MDA-MB231, JURKAT, and CEM cells, identified canonical pathways implicating these genes in kinase-mediated signal transduction (see green asterisks * ), including RAF-independent MAPK1/3 activation, RAF/MAP kinase cascade, Protein Kinase A Signaling, and the SAPK/JNK signaling pathway, as well as cell signaling processes (see blue asterisks * ), and membrane dynamics (see blue asterisks * ). The figure shown was recreated from the original IPA histogram to enlarge features and improve legibility.

Article Snippet: In addition, the RAMOS (ATCC, CRL-1596), CEM (ATCC, CCL-119), JURKAT (ATCC, TIB-152), NALM-6 (ATCC, CRL-3273), RPMI-8226, MM.1S, MM.1R, U266, Rec-1, JVM-13, A549 (ATCC, CRM-CCL-185), HCC70 (ATCC, CRL-2315), HCC1419 (ATCC, CRL-2326), T47D (ATCC, CRL-2865), KMS-11, and OVCAR-5 cell lines were grown in Roswell Park Memorial Institute-1640 (RPMI-1640) culture media (Cytiva-Hyclone, SH30027FS, UT, USA,) supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques: Transduction, Activation Assay, Membrane

Journal: Oncology Research

Article Title: Discovery of Two Novel Pyrazole Derivatives as Anticancer Agents Targeting Tubulin Polymerization and MAPK Signaling Pathways

doi: 10.32604/or.2026.074945

Figure Lengend Snippet: Connectivity map analyses revealed strong connectivity of P3C compounds with tubulin inhibitors. The top 20 perturbagens identified using P3C.1 and P3C.2 genes in common for ( A ) MDA-MB-231, and ( B ) Jurkat, and CEM cell lines. Tau scores of 90 or above have been previously recognized as strong and suitable for further investigation ( https://clue.io ).

Article Snippet: In addition, the RAMOS (ATCC, CRL-1596), CEM (ATCC, CCL-119), JURKAT (ATCC, TIB-152), NALM-6 (ATCC, CRL-3273), RPMI-8226, MM.1S, MM.1R, U266, Rec-1, JVM-13, A549 (ATCC, CRM-CCL-185), HCC70 (ATCC, CRL-2315), HCC1419 (ATCC, CRL-2326), T47D (ATCC, CRL-2865), KMS-11, and OVCAR-5 cell lines were grown in Roswell Park Memorial Institute-1640 (RPMI-1640) culture media (Cytiva-Hyclone, SH30027FS, UT, USA,) supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques:

Journal: PLoS Pathogens

Article Title: Reassessing the Role of APOBEC3G in Human Immunodeficiency Virus Type 1 Infection of Quiescent CD4+ T-Cells

doi: 10.1371/journal.ppat.1000342

Figure Lengend Snippet: (A) Quiescent CD4+ T-cells were purified from freshly isolated PBMCs of two independent donors with CD4 magnetic beads and nucleofected with siRNA targeting APOBEC3G (siA3G 883 ), conjugated with (FITC) or without FITC (None). Cells were analyzed by flow cytometry 3 hr after nucleofection. (B) To monitor the integrity of the RNAi machinery, quiescent CD4+ T-cells derived from PBMCs were nucleofected with siRNA targeting CD4 (siCD4), control siRNA (siControl), or no siRNA (None). The levels of cell surface CD4 expression were monitored by flow cytometry using PE-conjugated anti-CD4 antibody or isotype-matched control 48 hr after nucleofection and represented by mean fluorescent intensity (MFI) in each panel. The solid line represents PE-CD4 antibody stained cells, whereas the shaded area represents isotype control staining.

Article Snippet: After blocking with 5% skim milk in PBS with 0.05% Tween-20 (PBS-T), membranes were reacted with either polyclonal anti-APOBEC3G antibody (Cat. No. 10201, NIH AIDS Research and Reference Reagent Program) or polyclonal anti-β-Actin (Rockland Immunochemicals Inc., Gilbertsville, PA).

Techniques: Purification, Isolation, Magnetic Beads, Flow Cytometry, Derivative Assay, Control, Expressing, Staining

Journal: PLoS Pathogens

Article Title: Reassessing the Role of APOBEC3G in Human Immunodeficiency Virus Type 1 Infection of Quiescent CD4+ T-Cells

doi: 10.1371/journal.ppat.1000342

Figure Lengend Snippet: (A) Quiescent CD4+ T-cells derived from PBMCs were nucleofected with siRNAs and cultured for two days. Total RNA was isolated, and the levels of APOBEC3G mRNA were monitored by quantitative real time RT-PCR using β-actin as an internal control . P values (asterisks) versus control siRNA were 0.00022 (siA3G 240 WT ), 0.00013 (siA3G 726 ), 0.00005 (siA3G 883 ), respectively. (B) Quiescent CD4+ T-cells derived from PBMCs were nucleofected with siRNAs and cultured for two days. Cells were lysed in 0.5% SDS, and the levels of APOBEC3G protein were monitored by Western blotting. β-actin was used as a loading control.

Article Snippet: After blocking with 5% skim milk in PBS with 0.05% Tween-20 (PBS-T), membranes were reacted with either polyclonal anti-APOBEC3G antibody (Cat. No. 10201, NIH AIDS Research and Reference Reagent Program) or polyclonal anti-β-Actin (Rockland Immunochemicals Inc., Gilbertsville, PA).

Techniques: Derivative Assay, Cell Culture, Isolation, Quantitative RT-PCR, Control, Western Blot

Journal: Scientific Reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

doi: 10.1038/srep39100

Figure Lengend Snippet: (a) Mean and range of editing level at the 712 sites identified as targets for A3G-mediated editing are shown for the three A3G transfectant samples. The sites are ordered by the mean editing level. (b) Logo indicating sequence conservation and base frequency for sequences bearing the editing sites (at position 0). (c) Histogram of nucleotide lengths (b) of inverted repeat sequences flanking the editing sites.

Article Snippet: Sequence-verified plasmid constructs in pCMV6 vector for CMV promoter-driven expression of human A3G cDNA, with sequences matching NCBI RefSeq sequences NM_021822.1, for the generation of C-terminal Myc-DDK-tagged A3G proteins were obtained from OriGene (Rockville, MD; product number RC206821).

Techniques: Transfection, Sequencing

Journal: Scientific Reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

doi: 10.1038/srep39100

Figure Lengend Snippet: Gene features and effects on translation codon for A3G-mediated C> U RNA editing sites a .

Article Snippet: Sequence-verified plasmid constructs in pCMV6 vector for CMV promoter-driven expression of human A3G cDNA, with sequences matching NCBI RefSeq sequences NM_021822.1, for the generation of C-terminal Myc-DDK-tagged A3G proteins were obtained from OriGene (Rockville, MD; product number RC206821).

Techniques:

Journal: Scientific Reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

doi: 10.1038/srep39100

Figure Lengend Snippet: Sanger validation of selected C> U recoding RNA editing sites identified in 293T/A3G cells.

Article Snippet: Sequence-verified plasmid constructs in pCMV6 vector for CMV promoter-driven expression of human A3G cDNA, with sequences matching NCBI RefSeq sequences NM_021822.1, for the generation of C-terminal Myc-DDK-tagged A3G proteins were obtained from OriGene (Rockville, MD; product number RC206821).

Techniques:

Journal: Scientific Reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

doi: 10.1038/srep39100

Figure Lengend Snippet: Sanger chromatograms of cDNAs (in duplicate) and genomic DNAs (gDNA) (in triplicate) from control and A3G transfectants. Edited C is shaded black.

Article Snippet: Sequence-verified plasmid constructs in pCMV6 vector for CMV promoter-driven expression of human A3G cDNA, with sequences matching NCBI RefSeq sequences NM_021822.1, for the generation of C-terminal Myc-DDK-tagged A3G proteins were obtained from OriGene (Rockville, MD; product number RC206821).

Techniques:

Journal: Scientific Reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

doi: 10.1038/srep39100

Figure Lengend Snippet: (a) Immunoblot showing WT A3G expressed in whole cell lysate (20 μg) of 293T cells and recombinant (r) WT A3G (500 ng) obtained from 293T cells (purchased from Origene, Rockville, MD). Full-length blot is presented in (b) Cytidine deamination activity of recombinant A3G was examined in an in vitro reaction with a 5′ fluorescent dye-labeled ssDNA substrate of 40 nucleotides (left panel). Full-length gel is presented in . Sanger chromatograms of cDNAs of MED1 RNA from in vitro RNA editing assay containing rA3G in the presence of 100 nM (+) or 1 μM (++) and ART MED1 RNA (right panel). ( c ) Sanger chromatograms of cDNAs of A3G substrates ( MED1, KIAA1715, SCD, ITFG1, RFX7, GOLGA5, CHMP4B, CLASP1 ) (left panel) and A3A substrates ( VIM, ASCC2, TMEM179B, SDHB) (right panel) from in vitro RNA editing assay containing only rA3G or from in vitro RNA editing assay containing in vitro transcribed SDHB RNA and purified A3A protein ( SDHB lower panel). Edited cytidines are highlighted in black.

Article Snippet: Sequence-verified plasmid constructs in pCMV6 vector for CMV promoter-driven expression of human A3G cDNA, with sequences matching NCBI RefSeq sequences NM_021822.1, for the generation of C-terminal Myc-DDK-tagged A3G proteins were obtained from OriGene (Rockville, MD; product number RC206821).

Techniques: Western Blot, Recombinant, Activity Assay, In Vitro, Labeling, Purification

Journal: Scientific Reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

doi: 10.1038/srep39100

Figure Lengend Snippet: ( a ) Chromatograms of cDNAs (single) from ctrl./A3G transfected 293T cells shows the effect of mutations in A3G-NTD (W94A, C97S, W127A) and A3G-CTD (C291S) on C>U RNA editing (edited C is shaded black) in selected genes. ( b ) Bar graph showing RNA editing level of GOLGA5, KIAA1715 and MED1 in A3G-NTD and -CTD mutant transfectants. RNA editing levels (ratio of edited versus total RNA) are calculated by Sequencher TM software. The detection limit for relative height of minor peaks was 4.86% (depicted by a dotted line). Mean and SEM (n = 3) ( c ) Immunoblot showing A3G protein expression in whole cell lysates of 293T cells when transfected with empty vector (control), WT or various A3G-NTD and -CTD mutants. The D128K and P129A mutants were run on separate gels on the same day. The dashed line separates the two gels. Full-length blot is presented in ( d ) Bar graph depicting RNA editing level of KIAA1715, PRPSAP2, SCD and TM7SF3 cDNAs in WT or mutant transfectants. The detection limit is depicted by a dotted line. Mean and SEM (n = 3). Statistical analysis was performed by one-way ANOVA followed by multiple comparisons of RNA editing levels between WT and the mutants. Editing level is considered 0%, when the Sequencher software detects no secondary T peak. ****=adjusted P value ≤ 0.0001; NS = Not Significant.

Article Snippet: Sequence-verified plasmid constructs in pCMV6 vector for CMV promoter-driven expression of human A3G cDNA, with sequences matching NCBI RefSeq sequences NM_021822.1, for the generation of C-terminal Myc-DDK-tagged A3G proteins were obtained from OriGene (Rockville, MD; product number RC206821).

Techniques: Transfection, Mutagenesis, Software, Western Blot, Expressing, Plasmid Preparation

Journal: Scientific Reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

doi: 10.1038/srep39100

Figure Lengend Snippet: (a) Mean and range of editing level at the 712 sites identified as targets for A3G-mediated editing are shown for the three A3G transfectant samples. The sites are ordered by the mean editing level. (b) Logo indicating sequence conservation and base frequency for sequences bearing the editing sites (at position 0). (c) Histogram of nucleotide lengths (b) of inverted repeat sequences flanking the editing sites.

Article Snippet: Therefore, we purchased full length Myc-DDK tagged WT A3G protein from Origene (Rockville, MD) that was expressed in 293T cells and purified via affinity chromatography on anti-DDK column ( ).

Techniques: Transfection, Sequencing

Journal: Scientific Reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

doi: 10.1038/srep39100

Figure Lengend Snippet: Gene features and effects on translation codon for A3G-mediated C> U RNA editing sites a .

Article Snippet: Therefore, we purchased full length Myc-DDK tagged WT A3G protein from Origene (Rockville, MD) that was expressed in 293T cells and purified via affinity chromatography on anti-DDK column ( ).

Techniques:

Journal: Scientific Reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

doi: 10.1038/srep39100

Figure Lengend Snippet: Sanger validation of selected C> U recoding RNA editing sites identified in 293T/A3G cells.

Article Snippet: Therefore, we purchased full length Myc-DDK tagged WT A3G protein from Origene (Rockville, MD) that was expressed in 293T cells and purified via affinity chromatography on anti-DDK column ( ).

Techniques: Biomarker Discovery

Journal: Scientific Reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

doi: 10.1038/srep39100

Figure Lengend Snippet: Sanger chromatograms of cDNAs (in duplicate) and genomic DNAs (gDNA) (in triplicate) from control and A3G transfectants. Edited C is shaded black.

Article Snippet: Therefore, we purchased full length Myc-DDK tagged WT A3G protein from Origene (Rockville, MD) that was expressed in 293T cells and purified via affinity chromatography on anti-DDK column ( ).

Techniques: Control

Journal: Scientific Reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

doi: 10.1038/srep39100

Figure Lengend Snippet: (a) Immunoblot showing WT A3G expressed in whole cell lysate (20 μg) of 293T cells and recombinant (r) WT A3G (500 ng) obtained from 293T cells (purchased from Origene, Rockville, MD). Full-length blot is presented in (b) Cytidine deamination activity of recombinant A3G was examined in an in vitro reaction with a 5′ fluorescent dye-labeled ssDNA substrate of 40 nucleotides (left panel). Full-length gel is presented in . Sanger chromatograms of cDNAs of MED1 RNA from in vitro RNA editing assay containing rA3G in the presence of 100 nM (+) or 1 μM (++) and ART MED1 RNA (right panel). ( c ) Sanger chromatograms of cDNAs of A3G substrates ( MED1, KIAA1715, SCD, ITFG1, RFX7, GOLGA5, CHMP4B, CLASP1 ) (left panel) and A3A substrates ( VIM, ASCC2, TMEM179B, SDHB) (right panel) from in vitro RNA editing assay containing only rA3G or from in vitro RNA editing assay containing in vitro transcribed SDHB RNA and purified A3A protein ( SDHB lower panel). Edited cytidines are highlighted in black.

Article Snippet: Therefore, we purchased full length Myc-DDK tagged WT A3G protein from Origene (Rockville, MD) that was expressed in 293T cells and purified via affinity chromatography on anti-DDK column ( ).

Techniques: Western Blot, Recombinant, Activity Assay, In Vitro, Labeling, Purification

Journal: Scientific Reports

Article Title: The double-domain cytidine deaminase APOBEC3G is a cellular site-specific RNA editing enzyme

doi: 10.1038/srep39100

Figure Lengend Snippet: ( a ) Chromatograms of cDNAs (single) from ctrl./A3G transfected 293T cells shows the effect of mutations in A3G-NTD (W94A, C97S, W127A) and A3G-CTD (C291S) on C>U RNA editing (edited C is shaded black) in selected genes. ( b ) Bar graph showing RNA editing level of GOLGA5, KIAA1715 and MED1 in A3G-NTD and -CTD mutant transfectants. RNA editing levels (ratio of edited versus total RNA) are calculated by Sequencher TM software. The detection limit for relative height of minor peaks was 4.86% (depicted by a dotted line). Mean and SEM (n = 3) ( c ) Immunoblot showing A3G protein expression in whole cell lysates of 293T cells when transfected with empty vector (control), WT or various A3G-NTD and -CTD mutants. The D128K and P129A mutants were run on separate gels on the same day. The dashed line separates the two gels. Full-length blot is presented in ( d ) Bar graph depicting RNA editing level of KIAA1715, PRPSAP2, SCD and TM7SF3 cDNAs in WT or mutant transfectants. The detection limit is depicted by a dotted line. Mean and SEM (n = 3). Statistical analysis was performed by one-way ANOVA followed by multiple comparisons of RNA editing levels between WT and the mutants. Editing level is considered 0%, when the Sequencher software detects no secondary T peak. ****=adjusted P value ≤ 0.0001; NS = Not Significant.

Article Snippet: Therefore, we purchased full length Myc-DDK tagged WT A3G protein from Origene (Rockville, MD) that was expressed in 293T cells and purified via affinity chromatography on anti-DDK column ( ).

Techniques: Transfection, Mutagenesis, Software, Western Blot, Expressing, Plasmid Preparation, Control