Revision 1

#16029Store 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 (D1V5L) Rabbit mAb 50494 20 µl 33, 35 kDa Rabbit IgG
Phospho-STING (Ser365) (D8F4W) Rabbit mAb 72971 20 µl 41 kDa Rabbit IgG
cGAS (D3O8O) Rabbit mAb 31659 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 (D83B9) Rabbit mAb 4302 20 µl 45-55 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 Mouse-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 antibody 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.

    使用に関する制限

    法的な権限を与えられたCSTの担当者が署名した書面によって別途明示的に合意された場合を除き、 CST、その関連会社または代理店が提供する製品には以下の条件が適用されます。お客様が定める条件でここに定められた条件に含まれるものを超えるもの、 または、ここに定められた条件と異なるものは、法的な権限を与えられたCSTの担当者が別途書面にて受諾した場合を除き、拒絶され、 いかなる効力も効果も有しません。

    研究専用 (For Research Use Only) またはこれに類似する表示がされた製品は、 いかなる目的についても FDA または外国もしくは国内のその他の規制機関により承認、認可または許可を受けていません。 お客様は製品を診断もしくは治療目的で使用してはならず、また、製品に表示された内容に違反する方法で使用してはなりません。 CST が販売または使用許諾する製品は、エンドユーザーであるお客様に対し、使途を研究および開発のみに限定して提供されるものです。 診断、予防もしくは治療目的で製品を使用することまたは製品を再販売 (単独であるか他の製品等の一部であるかを問いません) もしくはその他の商業的利用の目的で購入することについては、CST から別途許諾を得る必要があります。 お客様は以下の事項を遵守しなければなりません。(a) CST の製品 (単独であるか他の資材と一緒であるかを問いません) を販売、使用許諾、貸与、寄付もしくはその他の態様で第三者に譲渡したり使用させたりしてはなりません。また、商用の製品を製造するために CST の製品を使用してはなりません。(b) 複製、改変、リバースエンジニアリング、逆コンパイル、 分解または他の方法により製品の構造または技術を解明しようとしてはなりません。また、 CST の製品またはサービスと競合する製品またはサービスを開発する目的で CST の製品を使用してはなりません。(c) CST の製品の商標、商号、ロゴ、特許または著作権に関する通知または表示を除去したり改変したりしてはなりません。(d) CST の製品をCST 製品販売条件(CST’s Product Terms of Sale) および該当する書面のみに従って使用しなければなりません。(e) CST の製品に関連してお客様が使用する第三者の製品またはサービスに関する使用許諾条件、 サービス提供条件またはこれに類する合意事項を遵守しなければなりません。

    Revision 1
    #16029

    Mouse-Reactive STING Pathway Antibody Sampler Kit

    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 1 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).
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from 3T3, Neuro-2a, and C2C12 cells using cGAS (D3O8O) Rabbit mAb.
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 3 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).
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from control HeLa cells (lane 1) or IRF-3 knockout HeLa cells (lane 2) using IRF-3 (D83B9) Rabbit mAb, #4302 (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.
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from various cell lines using STING (D1V5L) Rabbit mAb.
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 6 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).
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 7 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.
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from Raw 264.7 cells, untransfected (-) or transfected with poly(dA:dT) (5 μg/mL, 3 hr; +), using Phospho-STING (Ser365) (D8F4W) Rabbit mAb (upper), STING (D1V5L) Rabbit mAb (Rodent Preferred) #50494 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 9 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).
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 10 Expand Image
    Western Blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length mouse cGAS protein (mcGAS-Myc/DDK; +), using cGAS (D3O8O) Rabbit mAb.
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from various cell lines using TBK1/NAK (D1B4) Rabbit mAb.
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of extracts from COS-7 cells, mock transfected or transfected with human, mouse or rat IRF-3 constructs, using IRF-3 (D83B9) Rabbit mAb. The human and mouse constructs contain epitope tags.
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 13 Expand Image
    Immunoprecipitation of STING from Raw 264.7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is STING (D1V5L) Rabbit mAb. Western blot analysis was performed using STING (D1V5L) Rabbit mAb.
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 14 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).
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 15 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).
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 16 Expand Image
    Immunoprecipitation of cGAS from C2C12 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is cGAS (D3O8O) Rabbit mAb. Western blot analysis was performed using cGAS (D3O8O) Rabbit mAb.
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 17 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).
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 18 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.
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 19 Expand Image
    Western blot analysis of extracts from various cell lines using IRF-3 (D83B9) Rabbit mAb.
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 20 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.
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 21 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.
    Mouse-Reactive STING Pathway Antibody Sampler Kit: Image 22 Expand Image
    Western blot analysis of extracts from differentiated THP-1 cells, untreated or treated with LPS (1 µg/ml, 1 hour), using Phospho-IRF-3 (Ser396) (4D4G) Rabbit mAb #4947 (upper) or IRF-3 (D83B9) Rabbit mAb (lower).