Phospho irf3 antibody

Manufactured by Cell Signaling Technology

The Phospho-IRF3 antibody is a research-use only product that detects endogenous levels of IRF3 protein when phosphorylated at specific serine residues. It is a valuable tool for researchers studying interferon regulatory factor 3 (IRF3) signaling and activation.

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Lab products found in correlation

Tbk1 antibody, by Abcam (2 mentions) Anti flag m2 agarose, by Merck Group (2 mentions) Cgamp, by InvivoGen (2 mentions) β actin antibody, by Merck Group (2 mentions) Poly da dt, by Merck Group (1 mentions) Ezview red anti ha affinity gel, by Merck Group (1 mentions) Tubulin antibody, by Merck Group (1 mentions) Odyssey blocking buffer, by LI COR (1 mentions) Ifn α2a, by PBL Assay Science (1 mentions) Poly 1 c, by InvivoGen (1 mentions) Mouse monoclonal flag antibody, by Merck Group (1 mentions) Phospho tbk1, by Cell Signaling Technology (1 mentions) Irf3 antibody, by Santa Cruz Biotechnology (1 mentions) Herring testis dna, by Merck Group (1 mentions) Med1 antibody, by Fortis Life Sciences (1 mentions) Rnap 2 antibody, by Merck Group (1 mentions) Smc1a antibody, by Fortis Life Sciences (1 mentions)

4 protocols using phospho irf3 antibody

Generation and Characterization of Antibodies for cGAS and SENP7

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The polyclonal antibody against SENP7 was generated by immunizing rabbit with recombinant mouse SENP7 (716–1010 aa). The rabbit polyclonal antibody against cGAS was obtained by immunizing rabbit with recombinant mouse cGAS (362–643 aa). The antibodies against hemagglutinin (HA), Myc and IRF3 were purchased from Santa Cruz Biotechnology. Mouse monoclonal Flag antibody and β-actin antibodies were obtained from Sigma-Aldrich. The SUMO antibody was from Abcam. Phospho-IRF3 antibody, Phospho-TBK1 antibody, and rabbit DYKDDDDK tag antibody were from Cell Signaling Technology. TBK1 antibody was from Abcam. Anti-Flag (M2)-agarose and EZview red anti-HA affinity gel were from Sigma. Poly(dA:dT) was obtained from Sigma. Interferon stimulatory DNA (ISD) was prepared by annealing equimolar amounts of sense and antisense DNA oligonucleotides at 95°C for 10 min before cooling to room temperature. Oligonucleotides used are as follows: ISD (sense), 5′-TAC AGA TCT ACT AGT GAT CTA TGA CTG ATC TGT ACA TGA TCT ACA-3′; ISD (antisense), 5′-TGT AGA TCA TGT ACA GAT CAG TCA TAG ATC ACT AGT AGA TCT GTA-3′. cGAMP was from InvivoGen and was delivered into cultured cells by digitonin permeabilization method as previously described (Girardin et al., 2003).

Cui Y., Yu H., Zheng X., Peng R., Wang Q., Zhou Y., Wang R., Wang J., Qu B., Shen N., Guo Q., Liu X, & Wang C. (2017). SENP7 Potentiates cGAS Activation by Relieving SUMO-Mediated Inhibition of Cytosolic DNA Sensing. PLoS Pathogens, 13(1), e1006156.

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Immunoassays and Western Blotting Techniques

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Any immunofluorescence or IP-10 ELISAs were done as above.
Thp1-ISG-LUCIA, and RAW-ISG-LUCIA cell lines were obtained from Invivogen. LUCIA
assays were performed as described in manufacturer protocol. For Western blots,
phospho-IRF3 antibody was obtained from Cell Signaling (4947S) and tubulin
antibody was obtained from Sigma (T9026). We performed near-IR Western
blottoing, using Odyssey blocking buffer (LI-COR, P/N 927-40100) and following
LI-COR Odyssey near-IR Western blot protocol. Secondary antibodies were Goat
Anti-Rabbit 800 DyLight (Thermo, 35571) and Goat Anti-Mouse 680 DyLight (Thermo
35518), used at 1:15000 dilution.
For s. fig. 11,
THP1 cells were first differentiated using phorbol 12-myristate 13-acetate (PMA)
to make them more macrophage-like and adherent. Cells were treated with PMA (200
ng/mL) for approximately 18 hours, and media was subsequently washed out with
plain cell culture media.
Cell-titer-glo assay (s. fig. 10) was performed as per manufacturer protocol.

Koch P.D., Miller H., Yu G., Tallarico J., Sorger P.K., Wang Y., Feng Y., Thomas J.R., Ross N.T, & Mitchison T. (2018). A high content screen in macrophages identifies small molecule modulators of STING-IRF3 and NFkB signaling. ACS chemical biology, 13(4), 1066-1081.

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Characterization of cGAS Signaling Pathway

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The rabbit polyclonal antibody against cGAS was from Cell Signaling Technology and Sigma-Aldrich. The polyclonal antibody against RNF185 was from Abcam. The antibodies against hemagglutinin (HA), Myc, GFP, and ubiquitin were purchased from Santa Cruz Biotechnology. Mouse monoclonal Flag antibody and β-actin antibody were obtained from Sigma-Aldrich. The TBK1 antibody was from Abcam. The IRF3 antibody was from Santa Cruz Biotechnology. Phospho-TBK1 and Phospho-IRF3 antibody was from Cell Signaling Technology. The antibodies against CoxIV, Calreticulin and Calnexin were from Abcam. Anti-Flag (M2)-agarose was from Sigma-Aldrich.
Herring testis (HT) DNA was from Sigma. Salmon sperm DNA was from TREVIGEN. Poly(I:C) was purchased from Invivogen. IFNα2a was from PBL Assay Science. cGAMP was obtained from InvivoGen. In some experiments, cGAMP was delivered into cultured cells by digitonin permeabilization method as previously described [62 (link)].

Wang Q., Huang L., Hong Z., Lv Z., Mao Z., Tang Y., Kong X., Li S., Cui Y., Liu H., Zhang L., Zhang X., Jiang L., Wang C, & Zhou Q. (2017). The E3 ubiquitin ligase RNF185 facilitates the cGAS-mediated innate immune response. PLoS Pathogens, 13(3), e1006264.

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ChIP-qPCR Analysis of Transcription Factors

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ChIP was performed using standard protocols (38 (link)) on >1 × 10⁶ cells per immunoprecipitation, with an SMC1A antibody (Bethyl Labs, catalog # A300–055A), a Med1 antibody (Bethyl Labs, catalog # A300–793A), an RNAPII antibody (Millipore, catalog # 05–623) and a phospho-IRF3 antibody (Cell Signaling, catalog # 4947). See Supplementary Table S1 for primer sequences, designed with Primer 3 (39 (link)). Real-time PCR was performed as described above (see 3C), using 500 pg of ChIP DNA per reaction. If the starting sample size was ≥5, and the removal of a single data point reduced the standard error by over 20%, that data point was excluded as an ‘outlier.’ In these instances, no more than one data point was removed.

Banerjee A.R., Kim Y.J, & Kim T.H. (2014). A novel virus-inducible enhancer of the interferon-β gene with tightly linked promoter and enhancer activities. Nucleic Acids Research, 42(20), 12537-12554.

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