P tbk1 s172

Manufactured by Cell Signaling Technology

Sourced in United States

P-TBK1 S172 is a rabbit monoclonal antibody that recognizes TBK1 (TANK-binding kinase 1) phosphorylated at serine 172. TBK1 is a serine/threonine protein kinase that plays a role in the regulation of the immune response and inflammatory signaling pathways.

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7 protocols using p tbk1 s172

Western Blot Analysis of Cellular Proteins

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Cells were lysed using 2x LDS buffer containing 50mM DTT and boiled for 15 min. Approximately 50 μg of protein lysate was loaded onto 4%–12% Bis-Tris gels. Proteins were transferred onto PVDF membranes and blocked with 5% non-fat powdered milk. Primary antibodies were incubated 4°C overnight. Antibodies used: PINK1, p-ATM S1981, LC3B, TBK1, p-TBK1 S172, p-Histone H3 S10 and p-Histone H3 T11 (Cell Signaling), pATM S1981, COXII (Abcam), ATM (Cell Signaling or Sigma), Tim23 (BD Biosciences), Mfn2 (in-house), TOM20, Parkin, p53 (Santa Cruz), GAPDH and actin (Sigma), p62 (Cedarlane), and HA.11 (Covance). Secondary HRP-linked antibodies (GE Healthcare) were incubated at RT for 1 hr. Blots were exposed using peroxidase-based ECL (Pierce) detected by a ChemiDoc Imaging System (BioRad).

Sarraf S.A., Sideris D.P., Giagtzoglou N., Ni L., Kankel M.W., Sen A., Bochicchio L.E., Huang C.H., Nussenzweig S.C., Worley S.H., Morton P.D., Artavanis-Tsakonas S., Youle R.J, & Pickrell A.M. (2019). PINK1/Parkin Influences Cell Cycle by Sequestering TBK1 at Damaged Mitochondria, Inhibiting Mitosis. Cell reports, 29(1), 225-235.e5.

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Western Blot Analysis of Cellular Proteins

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Western Blot Analysis of Signaling Proteins

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Cell lysates were prepared in RIPA buffer (ThermoFisher Scientific) containing 1X protease inhibitor (Protease Inhibitor Cocktail, Roche) and phosphatase inhibitor (Roche, #4906837001) for 30 min on ice and then subjected to centrifugation at 14,000 rpm for 10 min. Protein concentration in the supernatants was measured with the Pierce BCA Protein Assay kit (ThermoFisher Scientific, #23227). 30 μg of protein was subjected to SDS-PAGE followed by transfer to nitrocellulose membranes (BIO-RAD, #1704270). Membranes were incubated with primary antibodies followed by rabbit or mouse antibodies conjugated with HRP or fluorescence dyes. Immunoblot images were taken using the Chemidoc MP imaging system (BIO-RAD). All antibodies were purchased from Cell Signaling Technology: cGAS (#83623), STING (#13647), p-TBK1 S172(#5483), TBK1 (#3504), p-IRF3 S386 (#37829), IRF3 (#11094), MYC (#9402), EZH2 (#4905), DNMT1 (#5032), KDM5B (#15327), KDM5C (#5361), and actin (#4970).

Lee K.M., Lin C.C., Servetto A., Bae J., Kandagatla V., Ye D., Kim G., Sudhan D.R., Mendiratta S., González Ericsson P.I., Balko J.M., Lee J., Barnes S., Malladi V.S., Tabrizi S., Reddy S.M., Yum S., Chang C.W., Hutchinson K.E., Yost S.E., Yuan Y., Chen Z.J., Fu Y.X., Hanker A.B, & Arteaga C.L. (2022). Epigenetic repression of STING by MYC promotes immune evasion and resistance to immune checkpoint inhibitors in triple negative breast cancer. Cancer immunology research, 10(7), 829-843.

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Immunoblotting Analysis of Tumor Signaling

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Protein lysates from tumor tissue and cultured tumor cells were made using RIPA buffer (8990, Thermo Fisher Scientific) containing protease and phosphatase inhibitors (88265 and A32959, respectively, Thermo Fisher Scientific). The following antibodies were used for immunoblotting: STING (13647S, Cell Signaling Technology), TBK1 (3504T, Cell Signaling Technology), p-TBK1 (s172) (5483T, Cell Signaling Technology), IRF3 (MA5-32348, Invitrogen, Thermo Fisher Scientific), p-IRF3 (4947S, Cell Signaling Technology), NF-κB (ab16502, Abcam), p–NF-κB (3033S, Cell Signaling Technology), PD-L1 (ab213480, Abcam, 66248-1-Ig, Proteintech), GAPDH (SC-32233, Santa Cruz Biotechnology), vinculin (4650S, Cell Signaling Technology), Foxp3 (ab215206, Abcam), and CTLA-4 (BE0164, Bio X Cell).

Somatilaka B.N., Madana L., Sadek A., Chen Z., Chandrasekaran S., McKay R.M, & Le L.Q. (2024). STING activation reprograms the microenvironment to sensitize NF1-related malignant peripheral nerve sheath tumors for immunotherapy. The Journal of Clinical Investigation, 134(10), e176748.

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Western Blot Analysis of Immune Signaling Proteins

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Tissues and cellular proteins were extracted using RIPA (Radio-Immunoprecipitation Assay) lysis buffer (Cell Signaling Technology, Danvers, Massachusetts, USA) supplemented with protease and phosphatase inhibitors (Cell Signaling Technology, Danvers, Massachusetts, USA). Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene 550 fluoride (PVDF) nitrocellulose membrane. Antibodies against MerTK, c-GAS, STING, phospho-STING (p-STING S365), TBK1, Phospho-TBK1 Ser172 (p-TBK1 S172), GAPDH (Cell Signaling Technology, Danvers, Massachusetts, USA), Cleaved caspase3 (Abcam, Branfold, Connecticut, USA), BCL-2, BAX (Proteintech, Rosemont, Illinois, USA), and ADAM17 (Novus Biologicals, Lehighton, Pennsylvania, USA) were used and incubated overnight at 4 °C. After 2 h of incubation with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Danvers, Massachusetts, USA), bands were detected using Immobilon ECL Ultra Western HRP substrate (Vazyme Biotechnology, Nanjing, China), and images were acquired using a Vilber chemiluminescent imaging system.

Hu H., Cheng X., Li F., Guan Z., Xu J., Wu D., Gao Y., Zhan X., Wang P., Zhou H., Rao Z, & Cheng F. (2023). Defective efferocytosis by aged macrophages promotes STING signaling mediated inflammatory liver injury. Cell Death Discovery, 9, 236.

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Synthesis and Utilization of cGAMP and Analogs

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2’3’-cyclic-GMP-AMP (cGAMP) was synthesized in house as described below. 3’3’-cyclic-GMP-AMP (3’3’-cGAMP), 3’3’-cyclic-di-AMP (3’3’-CDA), 3’3’-cyclic-di-GMP (3’3’-CDG), 2’3’-cyclic-di-AMP (2’3’-CDA), 2’3’-bisphosphothioate-cyclic-di-AMP (2’3’-CDA^S), and 2’3’-bisphosphothioate-cyclic-GMP-AMP (2’3’-cG^SA^SMP) were purchased from Invivogen. 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), sulfasalazine, folinic acid, and methotrexate were purchased from Sigma Aldrich. Sulfasalazine was dissolved in 50 mM NaHCO₃ and methotrexate was dissolved in 100 mM NaHCO₃. CellTiter-Glo luminescent cell viability assay was purchased from Promega. Rabbit polyclonal antibodies against TBK1 (1:1000), IRF3 (1:1000), pTBK1 (S172, 1:1000), pIRF3 (S396, 1:1000), AMPKa (1:1000), pAMPKa (THr172, 1:1000), STING (1:1000), and cGAS (1:1000) were purchased from Cell Signaling Technology. Mouse monoclonal anti-a-tubulin (1:1000) was purchased from Cell Signaling Technology.

Ritchie C., Cordova A.F., Hess G.T., Bassik M.C, & Li L. (2019). SLC19A1 is an importer of the immunotransmitter cGAMP. Molecular cell, 75(2), 372-381.e5.

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Inhibitors of Antiviral Signaling Pathways

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PKR inhibitor C16 (Tocris), IKKα:β inhibitor IKK16, TBK1 inhibitor Bx795 and JAK/STAT inhibitor Ruxolitinib (all Selleck Chemicals), and ISR inhibitor ISRIB (Sigma) were dissolved in DMSO and used at the indicated concentrations. Biotinylated HMW poly(I·C) was purchased from InvivoGen and recombinant IFNα2 was from PBL Assay Science. Primary antibodies used in this study were against eIF4G1, eIF4A, GAPDH, IRF3, p-IRF3(S396), STAT1, p-STAT1(Y701), IFNβ, IFIT1, PCBP2, PKR, ADAR, Dicer, IFI16, PACT, MDA5, TBK1, p-TBK1(S172), eIF2a, p-eIF2α(S51) (all Cell Signaling Technology), DHX9, LGP2, IFIT5 (all Proteintech), HelZ2, MCCC1 (ThermoFisher), DHX30 (Novus), NF90 (BD Biosciences), α-tubulin (Sigma-Aldrich), and p-PKR(T446) (LSBio). Anti-2C antibody was a gift from E. Wimmer (Stony Brook Univ., NY). Immunoblots were performed as described previously (59 (link), 60 (link)).

Dobrikov M.I., Dobrikova E.Y., McKay Z.P., Kastan J.P., Brown M.C, & Gromeier M. (2022). PKR Binds Enterovirus IRESs, Displaces Host Translation Factors, and Impairs Viral Translation to Enable Innate Antiviral Signaling. mBio, 13(3), e00854-22.

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