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43573
Type I Interferon Induction and Signaling Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Type I Interferon Induction and Signaling Antibody Sampler Kit #43573

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Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 1

Flow cytometric analysis of THP-1 cells differentiated with TPA #4174 (80 nM, 24 hr), and then untransfected (blue) or transfected with poly(dA:dT) (5 ug/mL, 3 hr; green), using Phospho-IRF-3 (Ser 386) (E7J8G) 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.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 2

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.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 3

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).

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 4

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).

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 5

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).

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 6

Western blot analysis of extracts from THP-1 cells, differentiated with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (80 nM, 16 hr) and then left untransfected (-) or transfected with poly(dA:dT) (5 μg/ml, 16 hr; +), using IFN-β1 (D1D7G) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). Brefeldin A #9972 (300 ng/ml) was added to cells 4 hr after the start of the transfection.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 7

Chromatin immunoprecipitations were performed with cross-linked chromatin from HT-1080 cells treated with IFN-γ (50 ng/ml) for 30 minutes and either Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human IRF-1 promoter primers, SimpleChIP® Human TAP1 Promoter Primers #5148, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 8

Western blot analysis of extracts from A549 cells (lane 1) or STAT1 knock-out cells (lane 2) using Stat1 (D1K9Y) Rabbit mAb #14994 (upper), and β-actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in the STAT1 knock-out A549 cells confirms specificity of the antibody for STAT1.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 9

Chromatin immunoprecipitations were performed with cross-linked chromatin from HT-1080 cells treated with Human Interferon-γ (hIFN-γ) #8901 (50 ng/ml, 30 min) and either Stat1 (D1K9Y) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human IRF-1 promoter primers, SimpleChIP® Human TAP1 Promoter Primers #5148, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 10

Chromatin immunoprecipitations were performed with cross-linked chromatin from U266 cells treated with Human Interferon-α1 (hIFN-α1) #8927 (10 nM, 30 min) and either IRF-9 (D2T8M) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human MX1 Promoter Primers #57949, human WARS intron 1 primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 11

Western blot analysis of extracts from HeLa cells, untreated (-) or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml, 16 hr; +), using IRF-9 (D2T8M) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 12

Immunohistochemical analysis of paraffin-embedded human ductal carcinoma of the breast using MX1 (D3W7I) Rabbit mAb performed on the Leica® Bond™ Rx.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 13

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.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 14

Confocal immunofluorescent analysis of THP-1 cells, untransfected (left) or transfected with Poly (I:C) (2.5 μg/ml, 6 hr; right), using Phospho-IRF-3 (Ser386) (E7J8G) XP® Rabbit mAb (green) and β-Actin (13E5) Rabbit mAb (Alexa Fluor® 647 Conjugate) #8584 (red). Blue = Hoechst 33342 #4082 (fluorescent DNA dye).

Type I Interferon Induction and Signaling Antibody Sampler Kit: 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.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 16

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.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 17

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 18

Flow cytometric analysis of ACHN cells using Stat1 (D1K9Y) Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 19

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml, 16 hr; right), using IRF-9 (D2T8M) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red).

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 20

Immunohistochemical analysis of paraffin-embedded T84 (left, positive) and HCT116 (right, negative) cell pellets using MX1 (D3W7I) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 21

Western blot analysis of extracts from A549 cells, untreated (-) or treated with Poly (I:C) (+), using Phospho-IRF-3 (Ser386) (E7J8G) XP® Rabbit mAb (upper) or IRF-3 (D6I4C) XP® Rabbit mAb #11904 (lower).

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 22

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

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 23

Immunohistochemical analysis of paraffin-embedded human lymphoma, control (left) or λ phosphatase-treated (right) using Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 24

Confocal immunofluorescent analysis of HeLa cells, serum-starved overnight (left) or treated with Human Interferon-α1 (hIFN-α1) #8927 (1,000 units/ml, 30 min; right), using Stat1 (D1K9Y) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red).

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 25

Immunoprecipitation of IRF-9 from THP-1 cell extracts. Lane 1 is 10% input, lane 2 is with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is with IRF-9 (D2T8M) Rabbit mAb. Western blot analysis was performed using IRF-9 (D2T8M) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 26

Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, untreated (left) or treated with Human Interferon-alpha1 (hIFN-alpha1) #8927 (right) using MX1 (D3W7I) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 27

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 28

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Stat1 (D1K9Y) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 29

Western blot analysis of extracts from various cell lines using IRF-9 (D2T8M) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 30

Immunohistochemical analysis of paraffin-embedded human non-small cell lung carcinoma using MX1 (D3W7I) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 31

Western blot analysis of extracts from various cell lines, untreated (-) or UV-treated (2 hr recovery; +), using Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 32

Immunohistochemical analysis of paraffin-embedded human lymphoma using Stat1 (D1K9Y) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 33

Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using MX1 (D3W7I) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 34

Western blot analysis of extracts from UV-treated HeLa cells, untreated (-) or CIP and λ phosphatase-treated (+), using Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb (upper) or Stat1 (42H3) Rabbit mAb #9175 (lower).

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 35

Immunoprecipitation of Stat1 from MCF7 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Stat1 (D1K9Y) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot was performed using Stat1 (9H2) Mouse mAb #9176.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 36

Immunohistochemical analysis of paraffin-embedded normal human spleen using MX1 (D3W7I) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 37

Western blot analysis of extracts from HeLa cells, untreated (-) or treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, 30 min; +), using Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb (upper) or Stat1 (42H3) Rabbit mAb #9175 (lower).

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 38

Western blot analysis of extracts from various cell lines using Stat1 (D1K9Y) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 39

Immunoprecipitation of MX1 from extracts of HeLa cells treated with Human Interferon-α1 #8927 (10 ng/ml, 16 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is MX1 (D3W7I) Rabbit mAb. Western blot analysis was performed using MX1 (D3W7I) Rabbit mAb.

Type I Interferon Induction and Signaling Antibody Sampler Kit: Image 40

Western blot analysis of extracts from HeLa cells, untreated (-) or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml, 16 hr; +), using MX1 (D3W7I) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

To Purchase # 43573T
Product # Size Price
43573T
1 Kit  (9 x 20 µl) $ 609

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-IRF-3 (Ser386) (E7J8G) XP® Rabbit mAb 37829 20 µl
  • WB
  • IF
  • F
H 50-55 Rabbit IgG
IRF-3 (D6I4C) XP® Rabbit mAb 11904 20 µl
  • WB
  • IP
  • IF
H Mk 50-55 Rabbit IgG
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
IFN-β1 (D1D7G) Rabbit mAb 73671 20 µl
  • WB
H 19, 21 Rabbit IgG
Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb 8826 20 µl
  • WB
  • IP
  • IHC
  • ChIP
H M R Mk 91 Rabbit IgG
Stat1 (D1K9Y) Rabbit mAb 14994 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H M R Mk 84, 91 Rabbit IgG
IRF-9 (D2T8M) Rabbit mAb 76684 20 µl
  • WB
  • IP
  • IF
  • ChIP
H 48 Rabbit IgG
MX1 (D3W7I) Rabbit mAb 37849 20 µl
  • WB
  • IP
  • IHC
H 76 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The Type I Interferon Induction and Signaling Antibody Sampler Kit provides an economical means of detecting the activation of pathways leading to upregulation of type I interferon (IFN), expression of IFN-β1, activation of signaling downstream of type I IFN, and expression of the MX1 interferon response gene, using phospho-specific and control antibodies. The kit includes enough antibodies to perform at least two western blot experiments.

Specificity / Sensitivity

Each antibody in the Type I Interferon Induction and Signaling Antibody Sampler Kit recognizes endogenous levels of its target protein. Phospho-IRF-3 (Ser386) (E7J8G) XP® Rabbit mAb recognizes endogenous levels of IRF-3 protein only when phosphorylated at Ser386. Phospho-IRF-7 (Ser477) (D7E1W) Rabbit mAb recognizes endogenous levels of IRF-7 protein only when phosphorylated at Ser477 and can also detect IRF-7 when dually phosphorylated at Ser477 and Ser479. Phospho-IRF-7 (Ser477) (D7E1W) Rabbit mAb may cross-react with unidentified proteins of 100 and 150 kDa. Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb recognizes endogenous levels of Stat1 protein only when phosphorylated at Ser727. Stat1 (D1K9Y) Rabbit mAb cross-reacts with an unidentified protein of 150 kDa. IRF-9 (D2T8M) Rabbit mAb cross-reacts with an unidentified protein of 95 kDa.

Source / Purification

Monoclonal antibodies are produced by immunizing rabbits with synthetic peptides corresponding to residues surrounding Pro115 of human IRF-7, Pro688 of human Stat1, Leu292 of human MX1, recombinant human IRF-3 protein, recombinant human IFN-β1 protein, and recombinant human IRF-9 protein. Phosphorylation-specific monoclonal antibodies are produced by immunizing rabbits with synthetic peptides corresponding to residues surrounding Ser386 of human IRF-3, Ser477 of human IRF-7, and Ser727 of human Stat1.

Background

The innate immune system uses pattern recognition receptors (PRRs) that detect conserved pathogen-associated molecular patterns (PAMPs), such as cytoplasmic double-stranded RNA, to detect and initiate an immune response to viral infection. Detection of virus by PRRs leads to phosphorylation and nuclear translocation of IRF-3 and IRF-7, resulting in upregulation of type I interferons, which include IFN-α and IFN-β (1-3). Type I interferons signal through the interferon α/β receptor (IFNAR), leading to phosphorylation and activation of Stat1 and Stat2, which form a complex with IRF-9 (4,5). This complex translocates to the nucleus where it induces transcription of interferon response genes including viral restriction factors, such as MX1, that limit viral replication and propagation (4-7).

  1. Servant, M.J. et al. (2003) J Biol Chem 278, 9441-7.
  2. Lin, R. et al. (2000) J Biol Chem 275, 34320-7.
  3. Sato, M. et al. (2000) Immunity 13, 539-48.
  4. Fu, X.Y. et al. (1990) Proc Natl Acad Sci U S A 87, 8555-9.
  5. Qureshi, S.A. et al. (1995) Proc Natl Acad Sci U S A 92, 3829-33.
  6. Staeheli, P. et al. (1986) Cell 44, 147-58.
  7. Staeheli, P. and Haller, O. (1985) Mol Cell Biol 5, 2150-3.

Pathways & Proteins

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Limited Uses

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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's 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's 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.

For Research Use Only. Not For Use In Diagnostic Procedures.
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