Rnase l

Manufactured by Abcam

Sourced in Hungary, France

RNase L is a cellular enzyme that functions as a key component of the innate immune response against viral infections. It is responsible for cleaving single-stranded RNA molecules, thereby inhibiting viral replication and propagation within the host cell.

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4 protocols using rnase l

Immunoblotting Analysis of Antiviral Proteins

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Samples were resolved on 4%, 7.5% or 12% SDS-PAGE and transferred to a nitrocellulose membrane using a semi-dry transfer unit (Bio-Rad). The membrane was incubated in blocking solution PBS-0.1% tween and 5% skimmed milk (Sigma) and immunoblotted with the following primary antibodies: FLAG (Sigma), RNAseL (Abcam), ZCCHV (for ZAP) (Abcam ab154680), OAS1 (Abcam ab86343) and OAS3 (Abcam ab154270). Antibody binding was detected by HRP-conjugated secondary antibodies followed by chemiluminescence detection by ECL prime western blotting reagent (Ge Healthcare). Images were analysed using Image software, and quantitative data were obtained for three independent experiments for ZAP upregulation.

Odon V., Fros J.J., Goonawardane N., Dietrich I., Ibrahim A., Alshaikhahmed K., Nguyen D, & Simmonds P. (2019). The role of ZAP and OAS3/RNAseL pathways in the attenuation of an RNA virus with elevated frequencies of CpG and UpA dinucleotides. Nucleic Acids Research, 47(15), 8061-8083.

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Antibody Sources for Protein Analysis

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Monoclonal E3L antiserum was a kind gift from S. Isaacs (Univ. Pennsylvania, PA). Polyclonal PABP1 antiserum was a kind gift of S. Morley (Univ. Sussex, UK). I3 antiserum was a kind gift of D. Evans (Univ. Alberta, Canada). All other antibodies were purchased commercially, as follows: Xrn1 (A300-443A; Bethyl Labs), α tubulin (6074; Sigma), eIF2α (5324; Cell Signaling), phospho(ser51)-eIF2α (3398; Cell Signaling), PKR (12297; Cell Signaling), phospho(T446)-PKR (32036; Abcam), PERK (5683; Cell Signaling), Hsc70 (10011384; Cayman Chemical); RNase L (13825; Abcam), dsRNA (J2; SciCons, Hungary). Phosphonoacetic acid (PAA) was from Sigma.

Burgess H.M, & Mohr I. (2015). Cellular 5′-3′ mRNA Exonuclease Xrn1 Controls Double-Stranded RNA Accumulation and Anti-Viral Responses. Cell Host & Microbe, 17(3), 332-344.

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Western Blot Protein Analysis

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Differentiated cells were washed and lysed in SDS-PAGE sample buffer (300 mM Tris (pH 8.9), 5% (W/V) SDS, 750 mM β-mercaptoethanol, 20% (V/V) glycerol, bromophenol blue). From these cellular extracts, proteins were fractionated, electrophoretically transferred onto nitrocellulose membrane to be then incubated with the different antibodies and analyzed with the Odyssey Infrared Imaging System LI-COR (Biosciences, ScienceTec, Courtabœuf, France) as previously described.^{29 (link)} We used primary antibodies against the following proteins: Phospho-Akt (Ser473), Akt, IκBα, PKR, Phospho-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, IRS1, SOCS3, COX IV (nine from Cell Signaling, Ozyme, Saint-Quentin Yvelines, France), RNase L (mouse), ABCE1 (mouse), OAS2 (three from Santa Cruz, Tebu-Bio, Le Perray en Yvelines, France), α-tubulin, UCP3 (both from Sigma-Aldrich), RNase L (human), Phospho-IRS1 (Ser312) (from Abcam, Paris, France), ABCE1 (human) (Abnova, Tebu-Bio) and MnSOD (Enzo Life Sciences, Villeurbanne, France). We used secondary antibodies conjugated to IRDye800 (Rockland, Tebu-Bio). α-Tubulin protein level was measured in each sample as an indicator of proteins quantity loading. Proteins levels were then quantified from membranes scans with the ImageJ software (http://rsbweb.nih.gov.gate2.inist.fr/ij/index.html) and corrected with the corresponding α-tubulin levels.

Fabre O., Breuker C., Amouzou C., Salehzada T., Kitzmann M., Mercier J, & Bisbal C. (2014). Defects in TLR3 expression and RNase L activation lead to decreased MnSOD expression and insulin resistance in muscle cells of obese people. Cell Death & Disease, 5(3), e1136-.

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Antibody Sources for Immunological Assays

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Monoclonal E3L antiserum was a kind gift from S. Isaacs (University of Pennsylvania). Polyclonal PABP1 antiserum was a kind gift of S. Morley (University of Sussex). I3 antiserum was a kind gift of D. Evans (University of Alberta). All other antibodies were purchased commercially as follows: Xrn1 (A300-443A; Bethyl Laboratories), α tubulin (6074; Sigma), eIF2α (5324; Cell Signaling Technology), phospho(ser51)-eIF2α (3398; Cell Signaling Technology), PKR (12297; Cell Signaling Technology), phospho(T446)-PKR (32036; Abcam), PERK (5683; Cell Signaling Technology), Hsc70 (10011384; Cayman Chemical), RNase L (13825; Abcam), dsRNA (J2; SciCons). PAA was from Sigma.

Burgess H.M, & Mohr I. (2015). Cellular 5′-3′ mRNA Exonuclease Xrn1 Controls Double-Stranded RNA Accumulation and Anti-Viral Responses. Cell Host & Microbe, 17(3), 332-344.

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