Revision 1

#38866Store at -20C

1 Kit

(7 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
STING (D2P2F) Rabbit mAb 13647 20 µl 33, 35 kDa Rabbit IgG
Phospho-STING (Ser366) (E9A9K) Rabbit mAb 50907 20 µl 40 kDa Rabbit IgG
cGAS (D1D3G) Rabbit mAb 15102 20 µl 62 kDa Rabbit IgG
TBK1/NAK (D1B4) Rabbit mAb 3504 20 µl 84 kDa Rabbit IgG
Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb 5483 20 µl 84 kDa Rabbit IgG
IRF-3 (D6I4C) XP® Rabbit mAb 11904 20 µl 50-55 kDa Rabbit IgG
Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb 29047 20 µl 45-55 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The Human-Reactive STING Pathway Antibody Sampler Kit provides an economical means of detecting activation and expression of key components of the STING pathway using phospho-specific and control antibodies. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

Stimulator of interferon genes (STING, TMEM173, MITA) is a transmembrane adaptor protein that is a critical component of the cellular innate immune response to pathogenic cytoplasmic DNA (1,2). STING is a ubiquitously expressed protein found predominantly in the ER (1). The enzyme cGAMP synthase (cGAS) produces the second messenger cyclic-GMP-AMP (cGAMP) in response to cytoplasmic DNA (3,4). cGAMP binds and activates STING (3,4). In addition, detection of cytoplasmic DNA by nucleic acid sensors, including DDX41 or IFI16, results in STING activation (5,6). Following activation, STING translocates with TBK1 to perinuclear endosomes and gets phosphorylated by ULK1 at Ser366 (Ser365 in mouse) (7,8). The TBK1 kinase phosphorylates and activates IRF-3 and NF-κB, which leads to the induction of type I interferon and other immune response genes (1,2,7).

  1. Ishikawa, H. and Barber, G.N. (2008) Nature 455, 674-8.
  2. Zhong, B. et al. (2008) Immunity 29, 538-50.
  3. Sun, L. et al. (2013) Science 339, 786-91.
  4. Wu, J. et al. (2013) Science 339, 826-30.
  5. Zhang, Z. et al. (2011) Nat Immunol 12, 959-65.
  6. Unterholzner, L. et al. (2010) Nat Immunol 11, 997-1004.
  7. Ishikawa, H. et al. (2009) Nature 461, 788-92.
  8. Konno, H. et al. (2013) Cell 155, 688-98.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #38866

    Human-Reactive STING Pathway Antibody Sampler Kit

    Human-Reactive STING Pathway Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from HeLa cells using IRF-3 (D6I4C) XP ® Rabbit mAb #11904. The virtual lane view (left) shows the target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (1 mg/ml) from cGAS (D1D3G) Rabbit mAb #15102. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from control HeLa cells (lane 1) or IRF-3 knockout HeLa cells (lane 2) using IRF-3 (D6I4C) XP® Rabbit mAb, #11904 (upper) or β-actin (13E5) Rabbit mAb, #4970 (lower). The absence of signal in the IRF-3 knockout HeLa cells confirms specificity of the antibody for IRF-3.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using STING (D2P2F) Rabbit mAb.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from various cell lines using cGAS (D1D3G) Rabbit mAb.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of adipocytes from wild type (WT) mice, untransfected (-) or transfected with Poly (I:C) (2.5 μg/ml, 6 hr; +), and adipocytes from IRF-3 (-/-) mice, untransfected (-) or transfected with Poly (I:C) (2.5 μg/ml, 6 hr; +), using Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb (upper), IRF-3 (D83B9) Rabbit mAb #4302 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of HCT116 cell extracts, untreated (-) or TBK1/NAK knock-out (+), using TBK/NAK (D1B4) Rabbit mAb Antibody #3504 (upper) or β-actin (D6A8) Rabbit mAb #8457 (lower).
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from THP-1 cells differentiated with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (80 nM, 16 hr) and then untransfected (-) or transfected with poly(dA:dT) (5 μg/mL, 3 hr; +) using Phospho-STING (Ser366) (E9A9K) Rabbit mAb (upper), STING (D2P2F) Rabbit mAb #13647 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight) followed by treatment with LPS (1 μg/ml), up to 24h, using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (upper), or total TBK1/NAK (D1B4) Rabbit mAb #3504 (lower).
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 10 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from various cell lines using IRF-3 (D6I4C) XP® Rabbit mAb.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-), transfected with a construct expressing human STING protein (hSTING; +), or transfected with a construct expressing mouse STING protein (mSTING; +), using STING (D2P2F) Rabbit mAb.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc-tagged full-length human cGAS protein (hcGAS-Myc; +), using cGAS (D1D3G) Rabbit mAb.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of HT-29 cells, untransfected (-) or transfected with Poly (I:C) (2.5 μg/ml, 6 hr; +), using Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb (upper) or IRF-3 (D6I4C) XP® Rabbit mAb #11904 (lower).
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 15 Expand Image
    Western blot analysis of extracts from various cell lines using TBK1/NAK (D1B4) Rabbit mAb.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 16 Expand Image
    Immunoprecipitation of Phospho-STING (Ser366) protein from THP-1 extracts differentiated with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (80 nM, 16 hr) and then transfected with poly(dA:dT) (5 ug/mL, 3 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-STING (Ser366) (E9A9K) Rabbit mAb. Western blot analysis was performed using Phospho-STING (Ser366) (E9A9K) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678, followed by Anti-mouse IgG, HRP-linked Antibody #7076 were used as the secondary antibodies.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 17 Expand Image
    Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight) followed by treatment with LPS (1 μg/ml, 1 hour), with or without phosphatase treatment using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (upper), or total TBK1/NAK (D1B4) Rabbit mAb #3504 (lower).
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 18 Expand Image
    Immunoprecipitation of IRF-3 from THP-1 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or IRF-3 (D6I4C) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using IRF-3 (D6I4C) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 and Anti-mouse IgG, HRP-linked Antibody #7076 were used as secondary antibodies.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 19 Expand Image
    Immunoprecipitation of STING from HL-60 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or STING (D2P2F) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using STING (D2P2F) Rabbit mAb.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 20 Expand Image
    Confocal immunofluorescent analysis of HT-29 cells, untransfected (left) or transfected with Poly (I:C) (2.5 μg/ml, 6 hr; right), using Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb (green) and EpCAM (VU1D9) Mouse mAb (Alexa Fluor® 555 Conjugate) #5488 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 21 Expand Image
    Confocal immunofluorescent analysis of THP-1 cells differentiated with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (80 nM, 16 hr) and then mock-transfected (left) or transfected with poly(dA:dT) (5 μg/mL, 3 hr; right) using Phospho-STING (Ser366) (E9A9K) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 22 Expand Image
    Confocal immunofluorescent analysis of THP-1 cells differentiated with TPA #4174 (80nM, overnight) (left), followed by treatment with LPS (1μg/ml, 1 hour) (center) or LPS with λ phosphatase treatment (right) using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 Phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 23 Expand Image
    Confocal immunofluorescent analysis of HT-29 cells, untransfected (left) or transfected with Poly(I:C) (2.5 μg/ml, 6 hr; right), using IRF-3 (D6I4C) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 24 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using STING (D2P2F) Rabbit mAb performed on the Leica® BOND™ Rx. 
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 25 Expand Image
    Flow cytometric analysis of adipocytes from wild type mice, untransfected (A) or transfected with Poly (I:C) (2.5 µg/ml, 6 hr; B), and adipocytes from IRF-3 (-/-) mice, untransfected (C) or transfected with Poly (I:C) (2.5 µg/ml, 6 hr; D), using Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 26 Expand Image
    Flow cytometric analysis of THP-1 cells differentiated with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (80 nM, 24 hrs), and then untransfected (blue) or transfected with poly(dA:dT) (5 ug/mL, 3 hr; green), using Phospho-STING (Ser366) (E9A9K) Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 27 Expand Image
    Flow cytometric analysis of THP-1 cells differentiated with TPA (80nM, 4 days) #9905, untreated (blue) or treated with LPS (1 ng/mL, 1 hr; green) #14011 using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded human neuroendocrine lung carcinoma using STING (D2P2F) Rabbit mAb performed on the Leica® BOND™ Rx. 
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 29 Expand Image
    Flow cytometric analysis of HT-29 cells, untransfected (blue) or transfected with Poly (I:C) (2.5 μg/ml, 6 hr; green), using Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 30 Expand Image
    Immunohistochemical analysis of paraffin-embedded human non-Hodgkin’s lymphoma using STING (D2P2F) Rabbit mAb performed on the Leica® BOND™ Rx. 
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 31 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma using STING (D2P2F) Rabbit mAb.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 32 Expand Image
    Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using STING (D2P2F) Rabbit mAb.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 33 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using STING (D2P2F) Rabbit mAb.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 34 Expand Image
    Immunohistochemical analysis of paraffin-embedded human tonsil using STING (D2P2F) Rabbit mAb.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 35 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse lung using STING (D2P2F) Rabbit mAb.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 36 Expand Image
    Immunohistochemical analysis of paraffin-embedded Syrian hamster uterus using STING (D2P2F) Rabbit mAb.
    Human-Reactive STING Pathway Antibody Sampler Kit: Image 37 Expand Image
    Immunohistochemical analysis of paraffin-embedded HT-29 (left, positive) and A549 (right, negative) cell pellets using STING (D2P2F) Rabbit mAb.
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.