Sting

Manufactured by Cell Signaling Technology

Sourced in United States

STING is a laboratory equipment product from Cell Signaling Technology. It functions as a sensor of cyclic dinucleotides, which are important signaling molecules in the innate immune response.

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Anti-STING antibody (10 citations) Phospho-STING (8 citations) Rabbit anti-STING antibody (4 citations) P-STING Ser366 (2 citations) Anti-human STING #13647 (2 citations) Rabbit monoclonal anti-STING (2 citations) Anti-STING rabbit polyclonal antibody (2 citations) Rabbit anti-human STING (2 citations) Phospho-STING (Ser365; D8F4W) Rabbit mAb (2 citations) Rabbit Phospho-STING (1 citations)

75 protocols using sting

Immunoblotting of Signaling Proteins

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MC38 tumor cells were washed with ice-cold PBS and harvested in radioimmunoprecipitation assay buffer (Sigma-Aldrich) supplemented with protease inhibitors, 10 mM NaF, and 4 mM Na₃VO₄ (Calbiochem). The lysates were clarified by centrifugation at 20,000g at 4°C, and the protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The proteins in 15 μg of sample were denatured with NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) for 10 min at 70°C, subsequently separated on a 10% polyacrylamide gel, transferred onto a nitrocellulose membrane (Cytiva), blocked with 5% nonfat dry milk in tris-buffered saline with Tween 20 (TBST) buffer (0.1% Tween 20) for 1 hour, and probed with the following primary and secondary antibodies: MLH1 (ab92312, Abcam), MSH2 (ab70270, Abcam), cGAS (#31659, Cell Signaling Technology), STING (#13647, Cell Signaling Technology), STING (#50494, Cell Signaling Technology), phospho-STAT1 (#8826, Cell Signaling Technology), phospho-STAT1 (#9167, Cell Signaling Technology), STAT1 (#9172, Cell Signaling Technology), HSP60 (BD Biosciences), anti-mouse IgG–horseradish peroxidase (HRP) (Cell Signaling Technology), and anti-rabbit IgG-HRP (Cell Signaling Technology). Visualization was performed by using Pierce ECL Western blotting substrates (Thermo Fisher Scientific).

Vornholz L., Isay S.E., Kurgyis Z., Strobl D.C., Loll P., Mosa M.H., Luecken M.D., Sterr M., Lickert H., Winter C., Greten F.R., Farin H.F., Theis F.J, & Ruland J. (2023). Synthetic enforcement of STING signaling in cancer cells appropriates the immune microenvironment for checkpoint inhibitor therapy. Science Advances, 9(11), eadd8564.

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Investigating DNA Damage Response Pathways

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ERI was from Fuji Film and PTX was from ADIPOGEN. The following antibodies were used for WB: cGAS (#15102S, 1:1000), STING (#13647, 1:1000), pIRF3 (#4947, 1:1000), Histone H3 (#9715, 1:1000), RAD51 (#D4B10, 1:1000), anti-rabbit IgG (#7074, 1:2000) and anti-mouse IgG (#7076, 1:2000) were from Cell Signaling Technology (CST), IFNβ (#PA5–20390, 1:1000) was from Invitrogen and Vinculin was from Santa cruz (#A1121, 1:1000). The following antibodies were used for IHC: STING (#13647, 1:400) was from Cell Signaling Technology and IFNβ (#PA5–20390, 1:400) was from Invitrogen. The following antibodies were used for IF: cGAS (#79978S, 1:500) was from CST, RAD51 (#ab133534, 1:500) was from abcam. IFNβ (#PA5–20390, 1:400) was from Invitrogen and LAP2 (#8197900, 1:1000) was from BD Biosciences. DNA Damage Detection Kit (#340–09431) was from Fuji Film.

Yamada H., Takada M., Ghone D., Yu M., Nagashima T., Fujimoto H., Sakakibara J., Hasegawa Y., Takao S., Yamada A., Narui K., Ishikawa T., Suzuki A, & Otsuka M. (2023). Eribulin induces micronuclei and enhances the nuclear localization of cGAS in triple-negative breast cancer cells. Research Square.

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Modulating cGAS-STING Signaling in Immune Cells

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cGAS agonist, G3-YSD (Invivogen, CA, USA) was reconstituted with water and transfected to cells using TransIT-LT1 transfection reagent (Mirus Bio, WI, USA). The following antibodies were used for immunohistochemistry: cGAS (#79978, 1:50), STING (#13647, 1:100), CD206 (#24595, 1:500), FOXP3 (#98377, 1:100), and αSMA (#19245, 1:600) from Cell Signaling Technology (Danvers, MA, USA); CD8a (#ab101500, 1:100) from Abcam (Cambridge, MA, USA). The following antibodies were used for immunoblot analysis: cGAS (#79978, 1:1000), STING (#13647, 1:1000), phospho-IRF3 (#29047, 1:1000), phospho-TBK1 (#5483, 1:1000), IRF3 (#11904, 1:1000), TBK1 (#3504, 1:1000), GAPDH (#2118, 1:1000) from Cell Signaling Technology.

Kabashima A., Matsuo Y., Ito S., Akiyama Y., Ishii T., Shimada S., Masamune A., Tanabe M, & Tanaka S. (2022). cGAS-STING signaling encourages immune cell overcoming of fibroblast barricades in pancreatic cancer. Scientific Reports, 12, 10466.

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Immunohistochemical and Immunofluorescence Analysis

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The paraffin sections were deparaffinized and subjected to antigen retrieval by steaming with sodium citrate buffer. mPTCs grown on collagen-coated coverslips were fixed with 4% paraformaldehyde. Primary antibodies for immunofluorescence included Ki67 (Cell Signaling Technology, 9129T), Bax (Abcam, ab182733), DLP1(BD Bioscience, 611112), Fis1 (Proteintech, 10956-1-AP) and dsDNA (Abcam,ab27156). Anti-rabbit or anti-goat or Alexa Fluor 647 conjugated secondary antibodies were used, and the fluorescent signals were visualized under a fluorescence microscope. Primary antibodies for immunohistochemical staining included KIM-1(R&D System, AF1750), PGC1 α (Novusbio, NBPI1-04676SS), COX I (Bioss, bs-3953R), STING(Cell Signaling Technology, 13647S),F4/80(Starter, S0B0227) and NGAL(R&D System, AF1757). The nucleus was counterstained with hematoxylin, dried, and mounted after being counterstained with DAB for color development. Using Image-Pro plus 6.0 software, positive staining regions were quantified.

Song Z., Xia Y., Shi L., Zha H., Huang J., Xiang X., Li H., Huang H., Yue R., Wang H, & Zhu J. (2024). Inhibition of Drp1- Fis1 interaction alleviates aberrant mitochondrial fragmentation and acute kidney injury. Cellular & Molecular Biology Letters, 29, 31.

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Evaluating STING Signaling Pathway

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Briefly, cells from pancreatic cell lines were washed twice with PBS and lysed in Pierce RIPA Buffer (Cat#8990, Thermo) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail 100×(Cat#78440, Thermo). Protein concentrations were quantified using Pierce BCA Protein Assay Kit (Cat#23225, Thermo). Samples were denatured at 95 °C in XT Sample Buffer 4×(Cat#1610791, Bio-Rad, Hercules, CA) and loaded onto 4–12% Criterion XT Bis-Tris Protein Gels (Cat#345-0124, Bio-Rad). Proteins were transferred onto PVDF Transfer Membrane (Cat#88518, Thermo) and probed for STING (Cat#13647S, Cell Signaling Technology, Danvers, MA), IRF3 (Cat#4302S, Cell Signaling), STAT1 (Cat#9172S, Cell Signaling), p-STAT1 (Y701) (Cat#7649S, Cell Signaling) and GAPDH (Cat#2118S, Cell Signaling). HRP-conjugated goat anti-rabbit IgG (Cat#31460, Invitrogen, Carlsbad, CA) was used as a secondary antibody. Proteins were visualized using SuperSignal West Pico PLUS Chemiluminescent Substrate (Cat#34580, Thermo).

Zebertavage L.K., Alice A., Crittenden M.R, & Gough M.J. (2020). Transcriptional Upregulation of NLRC5 by Radiation Drives STING- and Interferon-Independent MHC-I Expression on Cancer Cells and T Cell Cytotoxicity. Scientific Reports, 10, 7376.

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Investigating STING and IRF3 Signaling

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STING (Cell Signaling Technology Cat# 13647, RRID:AB_2732796), IRF3 (Cell Signaling Technology Cat# 11904, RRID:AB_2722521) and pIRF3(S396) (cat#29047) antibodies were purchased from Cell Signaling (Boston, MA, USA) and procedures for immunoblotting and immunofluorescence were performed as in our previous publication [3 (link)]. For R-loop immunofluorescence, the S9.6 antibody was purchased from Kerafast (Cat# ENH001, RRID:AB_2687463, Boston, MA, USA). The immunofluorescence protocol was modified, and the cells were fixed with 4% paraformaldehyde in PBS for 5 min and permeabilized with 0.01% saponin in PBS. Live staining with Sir-Hoechst (Spirochrome Inc, cat#SC007, Stein am Rhein, Switzerland) was performed following manufacturer instructions at 1 µM concentration in media. Picogreen staining was performed according to manufacturer instructions using Quant-iT^TM Picogreen^TM dsDNA Assay Kit (Cat#P7589. DAPI was purchased from Thermofisher Scientific (cat#62247, Waltham, MA, USA). For quantification, images were taken from 5 random positions across 2 slides of each treatment, positive cytoplasmic DNA staining was counted and representative images were shown. Three independent experiments were performed.

Cornelison R., Biswas K., Llaneza D.C., Harris A.R., Sosale N.G., Lazzara M.J, & Landen C.N. (2021). CX-5461 Treatment Leads to Cytosolic DNA-Mediated STING Activation in Ovarian Cancer. Cancers, 13(20), 5056.

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Protein Expression Analysis of Immune Signaling

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The hearts were instantly extracted and liquid nitrogen-frozen. RIPA lysis buffer (Beyotime Biotechnology, Nanjing, China) along with a cocktail of complete protease inhibitor were employed to isolate total protein from homogenized heart tissues. Following that, fractionation of 50 _g of extracted proteins was done on the 10% or 12% polyacrylamide gel and blotted onto PVDF membrane. Antibodies against cGAS (Cell Signaling Technology), STING (Cell Signaling Technology), TBK1 (Cell Signaling Technology), Phospho-TBK1 (Cell Signaling Technology), IRF3 (Cell Signaling Technology), Phospho-IRF3 (Cell Signaling Technology), NF-κB p65(Cell Signaling Technology), Phospho-NF-κB p65 (Cell Signaling Technology), Caspase-3 (Protein (Abcam) were adopted to assess protein expression via immunoblotting. The blots were then inoculated with radish peroxidase-linked rabbit secondary antibody immunoglobulin G (Cell Signaling Technology) after three washes. The loading control was GAPDH(Proteintech). Chemiluminescence was adopted to assess the protein bands, and a Bio-Rad with image software basic Quantity One was used to quantify them (Bio-Rad, Hercules, CA, USA).

Xiao Z., Yu Z., Chen C., Chen R, & Su Y. (2023). GAS-STING signaling plays an essential pathogenetic role in Doxorubicin-Induced Cardiotoxicity. BMC Pharmacology & Toxicology, 24, 19.

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Immunoblotting Analysis of Signaling Pathways

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Cells were lysed in RIPA lysis and extraction buffer (Thermo Fisher Scientific, Cat.#89900) containing 1xprotease inhibitors (Roche, Cat.#11-836-145-001) and phosphatase inhibitor (50 μM NaF and 100 μM Na₃VO₄). Immunoblotting was performed as described(33 (link)) using antibodies that specifically recognize MET (Cell Signaling Technology, Cat.#8198), Phospho-MET (Cell Signaling Technology, Cat.#3077), STING (Cell Signaling Technology, Cat.#13647), CD73 (Cell Signaling Technology, Cat.#13160), TBK1 (Cell Signaling Technology, Cat.#3013), Phospho-TBK1 (Cell Signaling Technology, Cat.#5483), IRF3 (Cell Signaling Technology, Cat.#11904), phospho-IRF3 (Cell Signaling Technology, Cat.#4947), STAT1 (Cell Signaling Technology, Cat.#9172), phosphor-STAT1 (Cell Signaling Technology, Cat.#9167), ERK(Cell Signaling Technology, Cat.#9107), phosphor-ERK (Cell Signaling Technology, Cat.#4370), AKT(Cell Signaling Technology, Cat.#9272), phosphor-AKT(Cell Signaling Technology, Cat.#4060), FRA1 (Abcam, Cat.#124722), phospho-FRA1 (Cell Signaling Technology, Cat.#5841), cGAS(Cell Signaling Technology, Cat.#15102), and β-actin (Cell Signaling Technology, Cat.#3700). Secondary antibodies were from LICOR Bioscience: IRDye 680LT Goat anti-Mouse IgG (#926–68020), IRDye 800CW Goat anti-Rabbit IgG (#926–32211). Imaging of blots are was performed using LICOR Odyssey system.

Yoshida R., Saigi M., Tani T., Springer B.F., Shibata H., Kitajima S., Mahadevan N.R., Campisi M., Kim W., Kobayashi Y., Thai T.C., Haratani K., Yamamoto Y., Sundararaman S.K., Knelson E.H., Vajdi A., Canadas I., Uppaluri R., Paweletz C.P., Miret J.J., Lizotte P.H., Gokhale P.C., Jänne P.A, & Barbie D.A. (2022). MET-Induced CD73 Restrains STING-Mediated Immunogenicity of EGFR-Mutant Lung Cancer. Cancer Research, 82(21), 4079-4092.

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Western Blot Analysis of PRC1 and STING

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Cell were lysed using RIPA buffer supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Total protein concentration was determined using BCA protein assay kit (ThermoFisher Scientific) and 10–20 μg of total protein were loaded per lane. Proteins were separated by electrophoresis on 4%–12% NuPAGE Bis-Tris Mini Gel (Invitrogen), and transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were incubated in primary antibodies at 4 °C overnight diluted as follows, BAP1 (Santa Cruz Biotechnology, sc-28383), 1:100; H2AK119Ub (Cell Signaling, 8240), 1:2000; RING1/RING1A (Cell Signaling, 2820), 1:250, (Origene, CF809319); RNF2/RING1B (Cell Signaling, 5694), 1:250, (Abcam, ab101273), 1:500; STING (Cell Signaling, 13647), 1:1000; 1:500; Actin (Cell Signaling, 4970), 1:2000. Corresponding HRP-conjugated secondary antibodies (mouse, Cell Signaling, 7076 and rabbit, Cell Signaling, 7074) were added and band intensities were visualized using the ChemiDoc MP Imaging system and Image Lab software (Bio-Rad).

Bakhoum M.F., Francis J.H., Agustinus A., Earlie E.M., Di Bona M., Abramson D.H., Duran M., Masilionis I., Molina E., Shoushtari A.N., Goldbaum M.H., Mischel P.S., Bakhoum S.F, & Laughney A.M. (2021). Loss of polycomb repressive complex 1 activity and chromosomal instability drive uveal melanoma progression. Nature Communications, 12, 5402.

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Tanreqing Injection Modulates Inflammatory Response

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Tanreqing injection (TRQ, 33 mg/ml) was provided by Shanghai Kaibao Pharmaceutical Company, China, Lot. No. 2003210. LPS (Escherichia coli 055: B5, L2880) was purchased from Sigma-Aldrich (St. Louis, MO, United States). Fetal bovine serum (FBS) was obtained from Invitrogen Gibco (Grand Island, NY). Penicillin and streptomycin, Dulbecco’s modified Eagle’s medium (DMEM), 0.25% trypsin, and phosphate buffer saline (PBS) were purchased from Meilunbio (Dalian, China). Antibodies against cGAS, P-STING, STING, P-TBK, TBK, P-IRF3, IRF3, NF-κB p65, P-P65, and P-IκBα were obtained from Cell Signaling Technology (Beverly, MA, United States). All the immunosorbent assay (ELISA) used in this study were acquired from Multiscience (Zhejiang, China), Elabscience (Wuhan, China), and Neobioscience Technology Company (Shenzhen, China). The MPO, LDH, MDA, GSH, and SOD assay kits were purchased from Nanjing Jiancheng Biology Institution (Nanjing, China). DNeasy Blood & Tissue Kit was purchased from Qiagen (Hilden, Germany). TB Green Premix Ex Taq II was acquired from TaKaRa (Beijing, China).

He Y.Q., Zhou C.C., Deng J.L., Wang L, & Chen W.S. (2021). Tanreqing Inhibits LPS-Induced Acute Lung Injury In Vivo and In Vitro Through Downregulating STING Signaling Pathway. Frontiers in Pharmacology, 12, 746964.

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