AGO1 | Argonaute 1
AS09 527 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana, Nicotiana benthamiana
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Chlamydomonas reinhardtii, Oryza sativa, Phaseolus vulgaris, Triticum aestivum, Zea mays
80 µg of Arabidopsis thaliana soluble total cell extract (extracted in 20 mMTris pH 7.5, 5mM MgCl2, 2.5mM DTT, 300 mM NaCl, 0.1% NP-40, 1% proteasome inhibitor MG132) was separated on 6% SDS-PAGE and blotted 1h to nitrocellulose. Filters were blocked 1h with 5% low-fat milk powder in TBS-TT (0.25% TWEEN20; 0.1% Triton-X) and probed with anti-AGO1 antibody (1:10 000, 1h) and secondary anti-rabbit (1:10000, 1 h) antibody (HRP conjugated) in TBS-TT containing 5% low fat milk powder. Antibody incubations were followed by washings in TBS-TT. All steps were performed at RT with agitation. Blots were developed for 5 min with ECL-PLUS detection reagent according to the manufacturer's instructions. Exposure time was 5 seconds.
Roche's protease inhibitor cocktail (no EDTA) can also be applied in extraction buffer.
Courtesy Dr. Ericka Havecker, University of Cambridge, UK
Reactant: Nicotiana benthamiana
Application: Western Blotting
Pudmed ID: 26641460
Journal: PLoS Pathog
Figure Number: 3C
Published Date: 2015-12-01
First Author: Hafrén, A., Lõhmus, A., et al.
Impact Factor: 6.506Open Publication
PGs are RNA granules to which also PVA RNA can localize.(A) Fractions containing PGs labeled with P0YFP were isolated from PVACPmut infected leaf lysates (+ sample), as described in materials and methods. Lysates from non-infected leaves expressing P0YFP were fractionated similarly (- sample). Three parallel fractions of each sample type were used for fluorescence quantification and relative fluorescence units are given as a mean ą standard deviation. (B) Fractions quantified in (A) were imaged using epifluorescence microscopy to show successful capture of PVA-induced PGs. Scale bar; 500 ?m. (C) PG fractions were isolated from mock-infiltrated leaves (-) and PVACPmut RNA-expressing leaves (+) at 3 DAI and subjected to a western blot detection of endogenous HCpro, P0 and AGO1. The asterisks denote the expected position of the corresponding protein. (D) Similar samples enriched for PGs as in (A and B), were incubated with or without RNAse A and imaged using epifluorescence microscopy. Scale bar; 20 ?m. (E) RNAse A-mediated release of fluorescence from isolated PGs (D) was quantified by analyzing fluorescence in total, soluble and low-speed pellet fractions after RNAse A treatment compared to control samples (n = 3). (F) PG fractions were prepared from leaves expressing PVA together with either P0YFP (control) or Strep-III-tagged P0 (P0SIII), and subjected to Strep-tag based affinity purification. RNA was isolated from the affinity-purified samples and subjected to reverse transcription (RT+), followed by PCR detection of viral RNA. Total RNA from PVA infected leaves was used as a positive control for RT-PCR. The RT-minus control (RT-) was negative. (G) Bacteriophage ? B-box RNA elements were fused to the 3ī UTR within PVA?GDD icDNA (PVAB-box; S1 Fig). Binding of ?N22RFP to the B-box RNA element enabled visualization of PVAB-box RNA in vivo. PGs were induced either by PVA?GDD (control) or PVAB-box and visualized by P0YFP (green channel), and ?N22RFP was co-expressed to label B-box RNA. The RFP signal was mainly found in nuclei due to the nuclear localization signal present in ?N22RFP, but also in the cytoplasm and PGs in the presence of PVAB-box (magenta channel). The images are projections of Z-stacks with a single layer inset from the area indicated with an arrow. Scale bar; 10 ?m.
Reactant: Arabidopsis thaliana (Thale cress)
Application: Western Blotting
Pudmed ID: 27938667
Figure Number: 5A
Published Date: 2016-12-12
First Author: Li, S., Le, B., et al.
Impact Factor: 7.448Open Publication
miRNAs and ta-siRNAs are recruited to membranes by AGO1.(A) Detection of three miRNAs in total extracts and the microsomal fraction in wild type (Col) and ago1-36. The ago1-36 mutant lacks the full-length AGO1 protein as shown by western blotting. HSC70 was a loading control. The three miRNAs were detected by northern blotting. 5S rRNA was an internal control and shown below each miRNA blot. The microsomal levels of the miRNAs were reduced in ago1-36. (B) A scatter plot showing comparable miRNA abundance in wild type (WT) and ago1-27 total extracts. Inset: box plots illustrating the distribution of miRNA RPMR values in WT and ago1-27. (C) A scatter plot showing that miRNAs have reduced microsomal enrichment in ago1-27 as compared with wild type. Microsomal enrichment was measured by M/T (ratio of levels in microsome vs. those in total extract). The degree of microsomal enrichment of miR390 (yellow dots) is largely unchanged, while that of most known ta-siRNA/phasiRNA triggers (red dots) is decreased in ago1-27. miR173’s (green dot) microsomal enrichment was weakly affected. (D) A scatter plot showing that 21-nt and 22-nt ta-siRNAs from TAS1-4 loci have reduced microsomal enrichment in ago1-27 as compared with wild type.DOI:http://dx.doi.org/10.7554/eLife.22750.010
Antibody binds microRNA and tasiRNAs, preference for 21nt miRNAs with 5'U.
To detect AGO1 in Nicotiana benthamiana, please inquire.
Recommended for detection of AGO1: extreme low femtogram range chemiluminescent detection reagent
TCA acetone precipitation method
AGO1 belongs to a group of argonaute proteins which are catalytic component of the RNA-incudes silencing complex (RISC). This protein complex is responsible for the gene silencing (RNAi).
Cabezas-Fuster et al. (2022). Missplicing suppressor alleles of Arabidopsis PRE-MRNA PROCESSING FACTOR 8 increase splicing fidelity by reducing the use of novel splice sites. Nucleic Acids Res. 2022 Jun 10;50(10):5513-5527. doi: 10.1093/nar/gkac338. PMID: 35639749; PMCID: PMC9177961.
Delenko et al. (2022) MicroRNA biogenesis and activity in plant cell dedifferentiation stimulated by cell wall removal. BMC Plant Biol. 2022 Jan 3;22(1):9. doi: 10.1186/s12870-021-03323-9. PMID: 34979922; PMCID: PMC8722089. (immunofluorescence)
Oliver & Martinez. (2021) Accumulation dynamics of ARGONAUTE proteins during meiosis in Arabidopsis. Plant Reprod. 2021 Nov 23. doi: 10.1007/s00497-021-00434-z. Epub ahead of print. PMID: 34812935.
Dalmadi et al. (2021) Controlled RISC loading efficiency of miR168 defined by miRNA duplex structure adjusts ARGONAUTE1 homeostasis. Nucleic Acids Res. 2021 Dec 16;49(22):12912-12928. doi: 10.1093/nar/gkab1138. PMID: 34850097; PMCID: PMC8682782.
Clavel et al. (2021) Atypical molecular features of RNA silencing against the phloem-restricted polerovirus TuYV. Nucleic Acids Res. 2021 Nov 8;49(19):11274-11293. doi: 10.1093/nar/gkab802. PMID: 34614168; PMCID: PMC8565345.
Brioudes et al. (2021) HASTY, the Arabidopsis EXPORTIN5 ortholog, regulates cell-to-cell and vascular microRNA movement. EMBO J. 2021 Aug 2;40(15):e107455. doi: 10.15252/embj.2020107455. Epub 2021 Jun 21. PMID: 34152631; PMCID: PMC8327949. (Immunoprecipitation)
Schwenk et al. (2021) Uncovering a novel function of the CCR4-NOT complex in phytochrome A-mediated light signalling in plants. Elife. 2021 Mar 30;10:e63697. doi: 10.7554/eLife.63697. PMID: 33783355; PMCID: PMC8009681.
Dunker, Lederer, and Weiberg. (2021). Plant ARGONAUTE Protein Immunopurification for Pathogen Cross Kingdom Small RNA Analysis. Bio-protocol 11(3): e3911. DOI: 10.21769/BioProtoc.3911.
Dunker et al. (2020). Oomycete small RNAs bind to the plant RNA-induced silencing complex for virulence. Elife. 2020 May 22;9:e56096.doi: 10.7554/eLife.56096.
Niedojadlo et al. (2020). Dynamic distribution of ARGONAUTE1 (AGO1) and ARGONAUTE4 (AGO4) in Hyacinthus orientalis L. pollen grains and pollen tubes growing in vitro. Protoplasma. 2020 Jan 8. doi: 10.1007/s00709-019-01463-2.
You et al. (2019). FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis. Nat Commun. 2019 Sep 27;10(1):4424. doi: 10.1038/s41467-019-12379-z
Dalmadi et al. (2019). AGO-unbound cytosolic pool of mature miRNAs in plant cells reveals a novel regulatory step at AGO1 loading. Nucleic Acids Res. 2019 Aug 8. pii: gkz690. doi: 10.1093/nar/gkz690.
Li (2019). The Isolation of Total and Membrane-Bound Polysomes from Arabidopsis and the Detection of Their Associated AGO1 and sRNAs. Methods Mol Biol. 2019;1932:317-333. doi: 10.1007/978-1-4939-9042-9_23.
Sprunck et al. (2019). Elucidating small RNA pathways in Arabidopsis thaliana egg cells. http://dx.doi.org/10.1101/525956
Chen et al. (2018). Structural and biochemical insights into small RNA 3' end trimming by Arabidopsis SDN1. Nat Commun. 2018 Sep 4;9(1):3585. doi: 10.1038/s41467-018-05942-7. (Immunoprecipitation)
Bologna et al. (2018). Nucleo-cytosolic Shuttling of ARGONAUTE1 Prompts a Revised Model of the Plant MicroRNA Pathway. Mol Cell. 2018 Feb 15;69(4):709-719.e5. doi: 10.1016/j.molcel.2018.01.007.
Zhang et al. (2017). RISC-interacting clearing 3'- 5' exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana. Elife. 2017 May 2;6. pii: e24466. doi: 10.7554/eLife.24466.
Schalk et al. (2017). Small RNA-mediated repair of UV-induced DNA lesions by the DNA DAMAGE-BINDING PROTEIN 2 and ARGONAUTE 1. Proc Natl Acad Sci U S A. 2017 Mar 21. pii: 201618834. doi: 10.1073/pnas.1618834114.
Dolata et al. (2016). Salt Stress Reveals a New Role for ARGONAUTE1 in miRNA Biogenesis at the Transcriptional and Posttranscriptional Levels. Plant Physiol. 2016 Sep;172(1):297-312. doi: 10.1104/pp.16.00830. (ChIP and Immunolocalization)
Pumplin et al. (2016). DNA Methylation Influences the Expression of DICER-LIKE4 Isoforms, Which Encode Proteins of Alternative Localization and Function. The Plant Cell November 14, 2016 tpc.00554.2016 . Advance Publication November 14, 2016.
Minoia et al. (2014). Specific Argonautes Selectively Bind Small RNAs Derived from Potato Spindle Tuber Viroid and Attenuate Viroid Accumulation In Vivo. J Virol. 2014 Oct 15;88(20):11933-45. doi: 10.1128/JVI.01404-14. Epub 2014 Aug 6.
Haveckeret al. (2010). The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell. 2010 Feb;22(2):321-34. doi: 10.1105/tpc.109.072199. (small-RNA-IP-Seq)
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