mouse monoclonal anti human ago2 antibody  (Bio-Techne corporation)

Journal: RSC Advances

Article Title: miR-183-96-182 clusters alleviated ox-LDL-induced vascular endothelial cell apoptosis in vitro by targeting FOXO1

doi: 10.1039/c8ra06866f

Figure Lengend Snippet: Upregulation of pro-apoptotic FOXO1 in ox-LDL treated HUVECs or HAECs is due to transcriptional regulation and reduction in the miRNA targeting mechanism. (A and B) Cell apoptosis in ox-LDL treated HUVECs or HAECs could be partially relieved by FOXO1 knockdown. (C–E) FOXO1 knockdown drastically reduced FOXO1 mRNA and protein expression levels in the HUVECs or HAECs with or without ox-LDL treatment. (F) FOXO1 promoter reporter assay suggests a mild upregulation of FOXO1 gene transcription. (G–J) AGO2-RIP assay shows that FOXO1 mRNA, miR-183-5p, miR-96-5p and miR-182-5p were downregulated in AGO2 co-immunoprecipitated complexes after ox-LDL treatment. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: A mouse monoclonal anti-human AGO2 antibody (H00027161-M01, Bio-techne) was incubated at 1 : 100 ratio with cell lysate for 2 hours at 4 °C with gentle agitation, followed by incubation with Pierce™ Protein A/G Magnetic Beads (Thermo Fisher Scientific) for another 2 hours at 4 °C with gentle agitation.

Techniques: Expressing, Reporter Assay, Immunoprecipitation

Journal: International journal of oncology

Article Title: Anisomycin inhibits angiogenesis in ovarian cancer by attenuating the molecular sponge effect of the lncRNA‑Meg3/miR‑421/PDGFRA axis.

doi: 10.3892/ijo.2019.4887

Figure Lengend Snippet: Figure 9. Anisomycin attenuates the molecular sponge effect in the lncRNA‑Meg3/miR‑421/PDGFRA axis. (A) Heatmap showing that the expression of lncRNA‑Meg3 in the anisomycin‑treated group was significantly lower than that of the control group. (B) Expression levels in clinical tumour samples and (C) in xenograft tumours suggest an inverse trend in expression for lncRNA‑Meg3 and miR‑421. (D) Expression levels of miR‑421 in HuOCSCs following anisomycin treatment. **P<0.01 vs. non‑treated (n=4). (E) Schematic of the complementary sites of mature miR‑421 and the 3’UTR of lncRNA‑Meg3 and PDGFRA mRNA. (F) Schematic of the structure of luciferase reporter plasmids. (G) The molecular sponge effect of lncRNA‑Meg3/miR‑421/PDGFRA. (H‑J) Results of the luciferase reporter assays. **P<0.01 vs. blank plasmid (n=3). (K) RIP results revealed that the binding between Meg3 and Ago2 decreased significantly following treatment of HuOCSCs with anisomycin. lncRNA, long non‑coding RNA; Meg3, maternally expressed 3; miR, microRNA; PDGFRA, platelet derived growth factor receptor α; HuOCSCs, human ovarian cancer stem cells; RIP, RNA immunoprecipitation; Ago2, argonaute 2; ORF, open reading frame; UTR, untranslated region; mut, mutant; IgG, immunoglobulin G.

Article Snippet: The mouse anti-human Ago2 monoclonal antibody (clone C34C6; cat no. 2897; Cell Signaling Technology, Inc.; 1:100) was added for 90 min, before the re-addition of 20 μg of protein A agarose beads to capture the immune complexes.

Techniques: Expressing, Control, Luciferase, Plasmid Preparation, Binding Assay, Derivative Assay, RNA Immunoprecipitation, Mutagenesis

Journal: PLoS ONE

Article Title: Identification of transforming growth factor-beta-regulated microRNAs and the microRNA-targetomes in primary lung fibroblasts

doi: 10.1371/journal.pone.0183815

Figure Lengend Snippet: (A) Western blot of Ago2 protein in the Ago2- and IgG-immunoprecipitation. M marker; T Total fraction; FT Flow through fraction; IP Immunoprecipitation fraction. Arrow indicates the Ago2 protein. Ago2 protein can be detected in the Ago2-IP fraction, while it cannot be detected in the IgG 1 -IP fraction. (B) To confirm miRNA enrichment in the IP fraction, qRT-PCR was performed for three randomly selected miRNAs expressed in lung fibroblasts, i.e. miR-31-5p, miR-455-3p and miR-199b. The levels of these miRNAs are strongly increased in the Ago2-IP fraction compared to the IgG 1 -IP fraction.

Article Snippet: Cell lysates were incubated overnight with monoclonal mouse anti-human Ago2 (Clone 2E12-1C9, Abnova, Taipei City, Taiwan) coated Sepharose G beads (Abcam, Cambridge, UK).

Techniques: Western Blot, Immunoprecipitation, Marker, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Identification of transforming growth factor-beta-regulated microRNAs and the microRNA-targetomes in primary lung fibroblasts

doi: 10.1371/journal.pone.0183815

Figure Lengend Snippet: For each miRNA, the percentages of predicted targets were calculated in all expressed genes and in the top 1,500 most IP-enriched genes in all four IP experiments. Chi-square test was used to determine whether the number of predicted targets in the Ago2-IP fraction for miR-455-3p and miR-21-3p in the top 1,500 most enriched genes was significant different from the expected based on the number of predicted targets in all expressed genes.

Article Snippet: Cell lysates were incubated overnight with monoclonal mouse anti-human Ago2 (Clone 2E12-1C9, Abnova, Taipei City, Taiwan) coated Sepharose G beads (Abcam, Cambridge, UK).

Techniques:

Journal: PLoS ONE

Article Title: Identification of transforming growth factor-beta-regulated microRNAs and the microRNA-targetomes in primary lung fibroblasts

doi: 10.1371/journal.pone.0183815

Figure Lengend Snippet: Most enriched biological processes/pathways within the Ago2-IP enriched and TargetScan predicted miR-455-3p target genes.

Article Snippet: Cell lysates were incubated overnight with monoclonal mouse anti-human Ago2 (Clone 2E12-1C9, Abnova, Taipei City, Taiwan) coated Sepharose G beads (Abcam, Cambridge, UK).

Techniques: Gene Expression, Activity Assay, Cell Differentiation, Activation Assay, In Utero, De-Phosphorylation Assay

Journal: PLoS ONE

Article Title: Identification of transforming growth factor-beta-regulated microRNAs and the microRNA-targetomes in primary lung fibroblasts

doi: 10.1371/journal.pone.0183815

Figure Lengend Snippet: Most enriched biological processes/pathways within the Ago2-IP enriched and TargetScan predicted miR-21-3p target genes.

Article Snippet: Cell lysates were incubated overnight with monoclonal mouse anti-human Ago2 (Clone 2E12-1C9, Abnova, Taipei City, Taiwan) coated Sepharose G beads (Abcam, Cambridge, UK).

Techniques: Activity Assay, Modification, Ubiquitin Proteomics, Phospho-proteomics, Activation Assay, Clinical Proteomics, Membrane, Starch