Revision 3

#52239Store at -20C

1 Kit

(9 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
Phospho-STING (Ser366) (D7C3S) Rabbit mAb 19781 20 µl 40 kDa Rabbit IgG
STING (D2P2F) Rabbit mAb 13647 20 µl 33, 35 kDa Rabbit IgG
Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb 29047 20 µl 45-55 kDa Rabbit IgG
IRF-3 (D6I4C) XP® Rabbit mAb 11904 20 µl 50-55 kDa Rabbit IgG
Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb 11927 20 µl 55 kDa Rabbit IgG
IRAK4 Antibody 4363 20 µl 55 kDa Rabbit 
Phospho-IRF-7 (Ser477) (D7E1W) Rabbit mAb 12390 20 µl 65 kDa Rabbit IgG
IRF-7 (D2A1J) Rabbit mAb 13014 20 µl 65 kDa Rabbit IgG
Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb 83186 20 µl 17 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 Innate Immunity Activation Antibody Sampler Kit provides an economical means of detecting the activation of multiple signaling pathways involved in innate immunity using phospho-specific, cleavage-specific, and control antibodies. The kit contains enough primary antibodies to perform at least two western blot experiments.

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 antibodies.

Background

The innate immune system responds rapidly to pathogens by detecting conserved pathogen-associated molecular patterns (PAMPs) and damage/danger-associated molecular patterns (DAMPs) through pattern recognition receptors (PRRs). There are several families of PRRs. Toll-like receptors (TLRs) are transmembrane PRRs and signal through recruitment of adaptor proteins, including MyD88, which leads to recruitment and phosphorylation of IRAK1 and IRAK4, followed by activation of NF-κB and MAP kinases (1-3). Some TLRs also activate IRFs, which upregulate the type I interferon response. Activation of TLR3 and TLR4 results in phosphorylation and activation of IRF-3, while TLR7, TLR8, and TLR9 lead to activation of IRF-7 (2, 3). STING is a multi-pass ER transmembrane protein that is activated in response to intracellular DNA downstream of DNA-sensing cytoplasmic PRRs, such as DDX41, or by binding the second messenger cyclic-GMP-AMP (cGAMP) produced by cGAS (4-6). Following activation, STING translocates with TBK1 to perinuclear endosomes, leading to phosphorylation and activation of IRF-3 and NF-κB (7, 8). Following activation and translocation, STING gets phosphorylated by ULK1, resulting in STING inactivation and degradation (9). Inflammasomes are cytoplasmic multimeric protein complexes that assemble in response to PAMPs or DAMPs detected by AIM2 or members of the nod-like receptor (NLR) family, such as NLRP3 (10). Inflammasomes activate Caspase-1, which cleaves the IL-1β and IL-18 precursor proteins into the mature forms (10).

  1. Janssens, S. and Beyaert, R. (2003) Mol Cell 11, 293-302.
  2. Barton, G.M. and Kagan, J.C. (2009) Nat Rev Immunol 9, 535-42.
  3. Blasius, A.L. and Beutler, B. (2010) Immunity 32, 305-15.
  4. Ishikawa, H. and Barber, G.N. (2008) Nature 455, 674-8.
  5. Zhang, Z. et al. (2011) Nat Immunol 12, 959-65.
  6. Sun, L. et al. (2013) Science 339, 786-91.
  7. Zhong, B. et al. (2008) Immunity 29, 538-50.
  8. Ishikawa, H. et al. (2009) Nature 461, 788-92.
  9. Konno, H. et al. (2013) Cell 155, 688-98.
  10. Rathinam, V.A. and Fitzgerald, K.A. (2016) Cell 165, 792-800.

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

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    Revision 3
    #52239

    Innate Immunity Activation Antibody Sampler Kit

    Innate Immunity Activation 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.
    Innate Immunity Activation Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from THP-1 cells using IRAK4 Antibody #4363. 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 separation module.
    Innate Immunity Activation 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.
    Innate Immunity Activation Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of serum-starved KARPAS-299 cell extracts, untreated (-) or treated with Human Interleukin-1β (hIL-1β) #8900 (50 ng/ml, 15 min; +), using Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb (upper) or IRAK4 Antibody #4363 (lower). Cell Line Source: Dr Abraham Karpas at the University of Cambridge.
    Innate Immunity Activation Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from HT-29 cells, untreated or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml, overnight) followed by transfection with poly(I:C) (2.5 μg/ml, 7 hr), as indicated, using Phospho-IRF-7 (Ser477) (D7E1W) Rabbit mAb (upper), IRF-7 Antibody #4920 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Innate Immunity Activation Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from HT-29 and G-361 cells, untreated (-) or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml, overnight; +), using IRF-7 (D2A1J) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Innate Immunity Activation Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from various cell lines using STING (D2P2F) Rabbit mAb.
    Innate Immunity Activation 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) (D7C3S) Rabbit mAb (upper), STING (D2P2F) Rabbit mAb #13647 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Innate Immunity Activation Antibody Sampler Kit: Image 9 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).
    Innate Immunity Activation Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from THP-1 (human), RAW 264.7 (mouse), and H-4-II-E (rat) cell lines, using IRAK4 Antibody.
    Innate Immunity Activation Antibody Sampler Kit: Image 11 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.
    Innate Immunity Activation Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of recombinant Human Interleukin-1β (hIL-1β) #8900 using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb.
    Innate Immunity Activation Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from various cell lines using IRF-3 (D6I4C) XP® Rabbit mAb.
    Innate Immunity Activation Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of extracts from HT-29 cells transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® IRF-7 siRNA I #13139 (+) or SignalSilence® IRF-7 siRNA II #13291 (+). Twenty-four hours after transfection, cells were treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml, overnight; +) and analyzed by western blot using IRF-7 (D2A1J) Rabbit mAb #13014 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The IRF-7 (D2A1J) Rabbit mAb confirms silencing of IRF-7 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.
    Innate Immunity Activation Antibody Sampler Kit: Image 15 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.
    Innate Immunity Activation Antibody Sampler Kit: Image 16 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).
    Innate Immunity Activation Antibody Sampler Kit: Image 17 Expand Image
    Western blot analysis of extracts from cells or media collected from THP-1 cells, differentiated with TPA #4147 (80 nM, overnight) and subsequently treated with (+) or without (-) Lipopolysaccharides (LPS) #14011 (1 μg/ml, 6 hr), using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb.
    Innate Immunity Activation 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.
    Innate Immunity Activation 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.
    Innate Immunity Activation 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).
    Innate Immunity Activation Antibody Sampler Kit: Image 21 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).
    Innate Immunity Activation Antibody Sampler Kit: Image 22 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using STING (D2P2F) Rabbit mAb performed on the Leica® BOND Rx. 
    Innate Immunity Activation Antibody Sampler Kit: Image 23 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.
    Innate Immunity Activation Antibody Sampler Kit: Image 24 Expand Image
    Confocal immunofluorescent analysis of THP-1 cells, differentiated with TPA #4174 (80 nM, 24 hr) and subsequently treated with (right) or without (left) Lipopolysaccharides (LPS) #14011 (1 μg/ml, 6 hr), using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Innate Immunity Activation Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded human neuroendocrine lung carcinoma using STING (D2P2F) Rabbit mAb performed on the Leica® BOND Rx. 
    Innate Immunity Activation Antibody Sampler Kit: Image 26 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.
    Innate Immunity Activation Antibody Sampler Kit: Image 27 Expand Image
    Immunohistochemical analysis of paraffin-embedded human non-Hodgkin’s lymphoma using STING (D2P2F) Rabbit mAb performed on the Leica® BOND Rx. 
    Innate Immunity Activation Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma using STING (D2P2F) Rabbit mAb.
    Innate Immunity Activation Antibody Sampler Kit: Image 29 Expand Image
    Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using STING (D2P2F) Rabbit mAb.
    Innate Immunity Activation Antibody Sampler Kit: Image 30 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using STING (D2P2F) Rabbit mAb.
    Innate Immunity Activation Antibody Sampler Kit: Image 31 Expand Image
    Immunohistochemical analysis of paraffin-embedded human tonsil using STING (D2P2F) Rabbit mAb.
    Innate Immunity Activation Antibody Sampler Kit: Image 32 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse lung using STING (D2P2F) Rabbit mAb.
    Innate Immunity Activation Antibody Sampler Kit: Image 33 Expand Image
    Immunohistochemical analysis of paraffin-embedded Syrian hamster uterus using STING (D2P2F) Rabbit mAb.
    Innate Immunity Activation Antibody Sampler Kit: Image 34 Expand Image
    Immunohistochemical analysis of paraffin-embedded HT-29 (left, positive) and A549 (right, negative) cell pellets using STING (D2P2F) Rabbit mAb.