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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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other Image 1 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 2 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 3 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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).

other Image 4 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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).

other Image 5 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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).

other Image 6 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 7 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 8 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 9 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 10 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 11 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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).

other Image 12 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 13 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 14 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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).

other Image 15 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 16 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 17 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 18 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 19 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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).

other Image 20 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 21 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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).

other Image 22 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 23 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 24 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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).

other Image 25 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 26 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 27 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 28 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 29 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 30 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 31 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 32 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 33 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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).

other Image 34 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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).

other Image 35 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 36 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 37 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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).

other Image 38 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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

other Image 39 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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.

other Image 40 - Type I Interferon Induction and Signaling Antibody Sampler Kit

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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