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52239
Innate Immunity Activation Antibody Sampler Kit

Innate Immunity Activation Antibody Sampler Kit #52239

Western Blotting Image 1

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 ug/mL, 3 hr) using Phospho-STING (Ser366) Antibody (upper), STING (D2P2F) Rabbit mAb #13647 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 2

Western blot analysis of extracts from various cell lines using STING (D2P2F) Rabbit mAb.

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Flow Cytometry Image 3

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.

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Western Blotting Image 4

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).

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Western Blotting Image 5

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).

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Western Blotting Image 6

Western blot analysis of extracts from various cell lines using IRF-3 (D6I4C) XP® Rabbit mAb.

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Western Blotting Image 7

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.

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Western Blotting Image 8

Western blot analysis of extracts from THP-1 (human), RAW 264.7 (mouse), and H-4-II-E (rat) cell lines, using IRAK4 Antibody.

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Western Blotting Image 9

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).

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Western Blotting Image 10

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).

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Western Blotting Image 11

Western blot analysis of recombinant Human Interleukin-1β (hIL-1β) #8900 using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb.

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Western Blotting Image 12

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.

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IP Image 13

Immunoprecipitation of phospho-STING (Ser366) from extracts of THP-1 cells 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 Normal Rabbit IgG #2729, and lane 3 is Phospho-STING (Ser366) Antibody. Western blot analysis was performed using Phospho-STING (Ser366) Antibody.

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Western Blotting Image 14

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.

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IP Image 15

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.

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Flow Cytometry Image 16

Flow cytometric analysis of KARPAS-299 cells, untreated (blue) or treated with Human Interleukin-1β (hIL-1β) #8900 (50 ng/ml, 15 min) (green), using Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody. Cell Line Source: Dr Abraham Karpas at the University of Cambridge.

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Western Blotting Image 17

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.

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Western Blotting Image 18

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.

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IP Image 19

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.

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Flow Cytometry Image 20

Flow cytometric analysis of HT-29 cells, untransfected (blue) or transfected with Poly (I:C) (2.5 μg/µl, 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.

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IF-IC Image 21

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).

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IP Image 22

Immunoprecipitation of Cleaved-IL-1β (Asp116) from extracts of THP-1 cells differentiated with TPA #4147 (80 nM, overnight) followed by treatment with Lipopolysaccharides (LPS) #14011 (1 μg/ml, 6 hr). Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is precipitated with Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb. Western blot was performed using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb.

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IHC-P (paraffin) Image 23

Immunohistochemical analysis of paraffin-embedded HT-29 (left, positive) and A549 (right, negative) cell pellets using STING (D2P2F) Rabbit mAb.

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IF-IC Image 24

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).

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IHC-P (paraffin) Image 25

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using STING (D2P2F) Rabbit mAb.

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IF-IC Image 26

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).

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IHC-P (paraffin) Image 27

Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using STING (D2P2F) Rabbit mAb.

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IHC-P (paraffin) Image 28

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using STING (D2P2F) Rabbit mAb.

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IHC-P (paraffin) Image 29

Immunohistochemical analysis of paraffin-embedded human tonsil using STING (D2P2F) Rabbit mAb.

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-STING (Ser366) Antibody 85735 20 µl
  • WB
  • IP
H 40, 48, 50 Rabbit 
STING (D2P2F) Rabbit mAb 13647 20 µl
  • WB
  • IP
  • IHC
H M 33, 35 Rabbit IgG
Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb 29047 20 µl
  • WB
  • IP
  • IF
  • F
H M R 45-55 Rabbit IgG
IRF-3 (D6I4C) XP® Rabbit mAb 11904 20 µl
  • WB
  • IP
  • IF
H Mk 50-55 Rabbit IgG
Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb 11927 20 µl
  • WB
  • IP
  • F
H 55 Rabbit IgG
IRAK4 Antibody 4363 20 µl
  • WB
  • IP
H M R Mk 55 Rabbit 
Phospho-IRF-7 (Ser477) (D7E1W) Rabbit mAb 12390 20 µl
  • WB
H 65 Rabbit IgG
IRF-7 (D2A1J) Rabbit mAb 13014 20 µl
  • WB
H 65 Rabbit IgG
Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb 83186 20 µl
  • WB
  • IP
  • IF
H 17 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

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.

Each antibody in the Innate Immunity Activation Antibody Sampler Kit detects endogenous levels of its target protein. Phospho-STING (Ser366) Antibody detects STING only when phosphorylated at Ser366. Phospho-IRF-3 (Ser396) (D6O1M) Rabbit mAb detects IRF-3 only when phosphorylated at Ser396. Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb detects IRAK4 only when phosphorylated at both Thr345 and Ser346 and does not react with IRAK4 when phosphorylated at only one of these sites. Phospho-IRF-7 (Ser477) (D7E1W) Rabbit mAb detects IRF-7 only when phosphorylated at Ser477. Phospho-IRF-7 (Ser477) (D7E1W) Rabbit mAb may cross-react with proteins of unknown origin between 100 and 150 kDa. Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb detects mature IL-1β only when cleaved at Asp116.

Monoclonal and polyclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Pro226 of STING, Lys41 of IRAK4, Pro115 of IRF-7, Asp116 of IL-1β, or recombinant human IRF-3 protein. Phospho-specific antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Ser366 of STING, Ser396 of IRF-3, Thr345/Ser346 of IRAK4, or Ser477 of IRF-7. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

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.
Entrez-Gene Id
3553 , 51135 , 3661 , 3665 , 340061
Swiss-Prot Acc.
P01584 , Q9NWZ3 , Q14653 , Q92985 , Q86WV6
For Research Use Only. Not For Use In Diagnostic Procedures.

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