Anti phospho tbk1 ser172

Manufactured by Cell Signaling Technology

The Anti-phospho-TBK1 (Ser172) is a lab equipment product that detects the phosphorylation of TBK1 at serine 172. It is a primary antibody that can be used in various immunoassays to identify and quantify the presence of phosphorylated TBK1 in biological samples.

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Anti-TBK1 (51 citations) Anti-phospho-TBK1 (26 citations) Phospho-TBK1 (23 citations) Phospho-TBK1 (Ser172) (11 citations) Rabbit anti-phospho-TBK1 (Ser172) (3 citations) Anti-phospho-TBK1/NAK (Ser172) (D52C2) (2 citations)

6 protocols using anti phospho tbk1 ser172

Western Blot and TB Protein Analysis

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For Western blot analysis, prepared WCL and nuclear fraction were denatured at 95°C for 10 min, separated by 12.5% SDS-PAGE gel. Proteins were transferred onto nitrocellulose membranes and blotted with rabbit anti-IRF3 (cat. A303-384A; Bethyl Laboratories Inc.), anti-IRF7 (cat. 3941; Prosci Inc.), anti-TBK1 (cat. 3504; Cell Signaling Technology), anti–phospho-TBK1 (Ser172; cat. 5483; Cell Signaling Technology), anti–RIG-I (cat. 3743; Cell Signaling Technology), anti–MDA-5 (cat. 5321; Cell Signaling Technology), anti-cGAS (cat. Sc-515777; SCBT), anti–β-actin (cat. 4970; Cell Signaling Technology), anti-MAVS (cat. Sc-365333; SCBT), anti-STING (cat. NBP2-24683SS), and anti-histone H3 (cat. 9717; Cell Signaling Technology) antibodies, followed by goat anti-rabbit IgG-HRP (cat. 31460; Thermo Scientific).
For M.tb culture supernatant proteins, M.tb strains were grown in Sauton’s liquid medium until midexponential phase, and culture supernatant fraction was prepared from 50 ml bacterial culture as described previously (Reyna et al., 2016 (link)). 10 µg of sample was loaded into 12% SDS-PAGE gel, and M.tb RNA polymerase subunit β (RNAP-β) and EsxB (CFP-10) proteins were probed using mouse anti–RNAP-β antibody (ab12087; Abcam) and rabbit anti-EsxB antibody (NR-13801; BEI Resources), respectively.

Cheng Y, & Schorey J.S. (2018). Mycobacterium tuberculosis–induced IFN-β production requires cytosolic DNA and RNA sensing pathways. The Journal of Experimental Medicine, 215(11), 2919-2935.

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Western Blot Analysis of Immune Signaling

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Lysates were prepared in lysis buffer (1x SDS Sample Buffer, 62.5 mM Tris-HCl pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue). Proteins were resolved by standard 10% SDS-PAGE and electroblotted onto nitrocellulose membranes. Membranes were blocked with 4% bovine serum albumin (w/v) in TBST and protein bands were detected with specific antibodies using chemiluminescence reagents and a G:BOX Chemi XRQ chemiluminescence imager (Syngene). The following rabbit antibodies were used: anti-phospho IRF3 (Ser 396) (1:1000; Cell Signaling #4947), anti-phospho-TBK1 (Ser 172) (1:1000; Cell Signaling #5483), and anti-ISG15 (1:1000; Cell Signaling #9636). Immunoreactive bands were visualized by incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulins (1:5000) or goat anti-mouse immunoglobulins (1:1000; Bio-Rad). To ensure that equal amounts of proteins were loaded, blots were re-probed with α-tubulin (1:3000; Sigma-Aldrich). To detect multiple proteins, membranes were re-probed after stripping of previously used antibodies using a pH 2.2 glycine-HCl/SDS buffer.

Ivin M., Dumigan A., de Vasconcelos F.N., Ebner F., Borroni M., Kavirayani A., Przybyszewska K.N., Ingram R.J., Lienenklaus S., Kalinke U., Stoiber D., Bengoechea J.A, & Kovarik P. (2017). Natural killer cell-intrinsic type I IFN signaling controls Klebsiella pneumoniae growth during lung infection. PLoS Pathogens, 13(11), e1006696.

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Western Blot Analysis of Immune Signaling

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Protein extracts from the BMDMs and BMDCs were run on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) Immobilon membranes (Thermo). Rabbit anti-STING, anti-cGAS, anti-phospho-IRF3 (Ser 396), anti-IRF3, anti-TBK1, anti-phospho-TBK1 (Ser 172), and horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies, all from Cell Signaling Technology; mouse monoclonal anti-DDX41 (Santa Cruz Biotechnology); and HRP-conjugated anti-mouse antibody (Sigma-Aldrich) were used for detection, using either ECL Western blot detection reagent or ECL prime Western blot detection reagent (GE Healthcare Life Sciences).

Stavrou S., Aguilera A.N., Blouch K, & Ross S.R. (2018). DDX41 Recognizes RNA/DNA Retroviral Reverse Transcripts and Is Critical for In Vivo Control of Murine Leukemia Virus Infection. mBio, 9(3), e00923-18.

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Immunoblotting and Immunofluorescence Antibody Dilutions

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Antibodies used for immunoblotting and their corresponding dilutions were anti-MAVS (Santa Cruz Biotech sc-365334; 1:500), anti-TOM20 (Santa Cruz Biotech sc-11415; 1:300), anti-alpha Sr-1 (Abcam ab28052; 1:1,500), anti-β-actin (Santa Cruz Biotech sc-1615-hrp, 1:2,000), anti-MFN2 (Cell Signaling #9482; 1:800), anti-TBK1 (Cell Signaling #3504; 1:1,000), anti-phospho-TBK1 (Ser172) (Cell Signaling #5483; 1:700), goat anti-rabbit IgG-HRP (Millipore #12-348; 1:2000), and goat anti-mouse IgG-HRP (Millipore #12-349; 1:2,000). Primary antibodies used for immunofluorescence were anti-MAVS (Santa Cruz Biotech sc-365334; 1:100 dilution), anti-TOM20 (Abcam ab56783; 1:100 dilution), anti-FACL4 (Thermo PA5-27137; 1:50), anti-PMP70 (Abcam ab3424; 1:650), anti-MFN2 (Cell Signaling #9482; 1:50), anti-TRAF3 (Santa Cruz Biotech sc-1828or Thermo Fisher Scientific PA1-41107; 1:50), anti-alpha Sr-1 (Abcam ab28052; 1:50), anti-vimentin (Thermo Fisher Scientific PA1-16759; 1:2,000), anti-GM130 (BD Biosciences #610822; 1:250), anti-reovirus σ1 5C6 and G5 ((95 (link)); mix of 1:2,500 each; Fig. 6B), and rabbit anti-reovirus T3D antisera (B. Sherry, unpublished; 1:2,000; Fig S5). Secondary antibodies were Alexa^® 488-, Alexa^® 594- or Alexa^® 647-conjugated goat anti-mouse, anti-rabbit or anti-chicken IgG (Thermo Fisher Scientific; 1:750 - 1:1,000).

Rivera-Serrano E.E., DeAngelis N, & Sherry B. (2017). Spontaneous Activation of a MAVS-dependent Antiviral Signaling Pathway Determines High Basal Interferon-β Expression in Cardiac Myocytes. Journal of molecular and cellular cardiology, 111, 102-113.

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Immunoblot Analysis of Signaling Proteins

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WCL and nuclear fraction were denatured at 95°C for 10 min and separated by 12.0% SDS-PAGE gel. Proteins were transferred onto nitrocellulose membranes and probed with anti-IRF3 (Cat.A303-384A, Bethyl Laboratories Inc), anti-IRF7 (Cat. 3941; Prosci Inc.), anti-TBK1 (Cat.3504, Cell Signaling Technology), anti-phospho-TBK1 (Ser172) (Cat.5483, Cell Signaling Technology), anti-NF-κB (Cat. 8242T, Cell Signaling Technology), anti-Ets-2 (cat. sc-365666, SCBT), anti-ETV5 (Cat. sc-100941, SCBT), anti-Stat1 (Cat. sc-591, SCBT), anti-AP-2(Cat. sc-12726, SCBT), anti-SP1(Cat. sc-17824, SCBT), anti-iNOS (Cat. 610599, BD Bioscience), anti-β-actin (Cat.4970, Cell Signaling Technology), and anti-Histone H3 (Cat.9717, Cell Signaling Technology) antibodies, followed by goat anti-rabbit IgG-HRP (Cat.31460, Thermo Scientific) or goat anti-mouse IgG-HRP (Cat.31438, Thermo Scientific).

Cheng Y., Kiene N.J., Tatarian A., Eix E.F, & Schorey J.S. (2020). Host cytosolic RNA sensing pathway promotes T Lymphocyte-mediated mycobacterial killing in macrophages. PLoS Pathogens, 16(5), e1008569.

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Western Blot Protein Detection

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Following protein transfer, nitrocellulose membranes were probed with protein-specific primary antibodies diluted in PBS containing 5% Milk or 5% BSA as follows: anti-actin Cat #A5441 from Sigma Aldrich; anti-tubulin Cat #5286 from Santa Cruz; anti-Flag M2 Cat #SLBF6631V from Sigma Aldrich; anti-Phospho-STING (Ser 366 ) Cat #19781 from Cell signaling; anti-STING Cat #13647 from Cell signaling; anti-Phospho-TBK1 (Ser 172 ) Cat #5483 from Cell signaling; anti-TBK1/IKKε Cat #IMG270A from Imgenex. Membranes were washed using dPBS containing 0.5% Tween (PBS-T) before incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Seracare or Jackson Immunoresearch Laboratories). After final wa shes, enhanced chemiluminescence using the Western Lightning Chemiluminescence Reagent Plus (Perkin-Elmer Life Sciences) was performed to identify immunoreactive bands detected using a LAS4000mini CCD camera apparatus (GE healthcare).

Cuervo N.Z., Fortin A., Caron E., Chartier S., & Grandvaux N. (2020). Pinpointing of cysteine oxidation sites in vivo by high-resolution proteomics reveals mechanism of redox-dependent inhibition of STING.

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