Comprehensive Immunoblotting Assay for RIG-I-Like Receptor Signaling

RNA polymerase III inhibitor (ML-60218) was from Merck Millipore. MDA5 (D74E4, rabbit mAb #5321), STING (antibody #3337), phospho-IRF-3 (Ser396) (4D4G, rabbit mAb #4947), IRF-3 (D6I4C, XP rabbit mAb #11904), RIG-I (D14G6, rabbit mAb #3743), MAVS (antibody #3993), TBK1/NAK (D1B4, rabbit mAb #3504), phospho-TBK1/NAK (Ser 172) (D52C2, rabbit mAb, #5483), IKKε (D20G4, rabbit mAb, #2905), cGAS (E9G9G, rabbit mAb, #83623), beta actin HRP conjugate (13E5, rabbit mAb # 5125), anti-rabbit IgG HRP-linked (antibody #7074), histone H2AX (antibody #2595), antibodies were from Cell Signaling Technology. GAPDH (mouse mAb, #G8795) was from Sigma Aldrich. Anti-mouse IgG HRP-linked (antibody #NA931V) was from GE Healthcare. Monoclonal anti-β-actin-peroxidase (antibody #A3854) and digitonin (D141) were from Sigma Aldrich. Anti-rabbit MEK1/2 (#8727), anti-rabbit AIF (#5318) and anti-rabbit Histone H3 (#4499) were from cell signaling, anti-rabbit ALAS1 (#MA5-35584) were from Invitrogen. TURBO DNase (2U/μl, #AM2238) was from Invitrogen. GlycoBlue Coprecipitant (AM9515) was from Applied Biosystems. TOM20 (EPR15581-39, rabbit mAb, #ab186734) and mCherry (1C51, rabbit mAb, #ab125096) were from Abcam. RIG-I (D33H10, rabbit mAb, #4200) and mouse anti-rabbit IgG (conformation specific, L27A9, mAb #3678), cell lysis buffer (#9803), protein A magnetic beads (#73778), glycine (#7005) and PMSF (#8553) were from Cell Signaling. Recombinant RNasin Ribonuclease inhibitor (#N2515) was from Promega. Secondary Alexa Fluor 488 goat anti-mouse (#A11029), Alexa Fluor 488 goat anti-rabbit (#A11034), Alexa Fluor 633 goat anti-mouse (#A21053), Alexa Fluor 647 goat anti-rabbit (#A21244), Fluoromount-G with DAPI (#00-4959-52) and SlowFade Diamond Antifade Mountant without DAPI (#S36972) were from Invitrogen. DAPI solution 1.0 mg/ml (564907) was from BD Pharmingen. Monoclonal Alexa Fluor 647 Mouse anti-H2AX (pS139) (#560447) was from BD Pharmingen. Annexin V Apoptosis Detection kit (888007) with Annexin V efluor 450 (48-8006-69) and Fixable viability Dye eFluor 780 (65-0865-14) were from eBioscience. JetPRIME (#114–07) was from Polyplus Transfection and Lipofectamine RNAiMAX (#13778075) was from Invitrogen. NucleoSpin Gel and PCR Clean-up kit (#740609) was from Macherey-Nagel. Quant-iT dsDNA Assay kit, High sensibility (#Q33120) was from Invitrogen and NEBNext DNA Library Prep kit (#E7645) and NEBNext Multiplex Oligos for Illumina (#E7600) were from New England BioLabs.

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Khalfi P., Suspène R., Raymond K.A., Caval V., Caignard G., Berry N., Thiers V., Combredet C., Rufie C., Rigaud S., Ghozlane A., Volant S., Komarova A.V., Tangy F, & Vartanian J.P. (2023). Antagonism of ALAS1 by the Measles Virus V protein contributes to degradation of the mitochondrial network and promotes interferon response. PLOS Pathogens, 19(2), e1011170.

Publication 2023

Actin Alexa fluor 488 Alexa fluor 647 Annexin v Anti igg Antibodies Antibody Apoptosis Assay Beta actin Buffer Cell Cgas Dapi Diamond Digitonin Dna library Dnase Dsdna Gapdh Glycine Goat Histone h2ax Histone h3 Irf 3 Lipofectamine Mavs Mda5 Mek1 Mouse Oligos Peroxidase Promega Protein a Rabbit Ribonuclease Rig i Rna polymerase iii Transfection

Corresponding Organization : Department of Virology

Other organizations : Sorbonne Université, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement, Agence Nationale de Sécurité Sanitaire de l’Alimentation, de l’Environnement et du Travail, NeuroDiderot, École Nationale Vétérinaire d'Alfort

Top 5 similar protocols

Variable analysis

independent variables

ML-60218 (RNA polymerase III inhibitor) from Merck Millipore

dependent variables

Protein expression levels of MDA5, STING, phospho-IRF-3, IRF-3, RIG-I, MAVS, TBK1/NAK, phospho-TBK1/NAK, IKKε, cGAS, β-actin, GAPDH, MEK1/2, AIF, Histone H3, ALAS1, H2AX

control variables

β-actin HRP conjugate
Anti-rabbit IgG HRP-linked
Anti-mouse IgG HRP-linked
Monoclonal anti-β-actin-peroxidase
GAPDH
MEK1/2
Histone H3
ALAS1

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