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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

Citations (0)
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.
CUT&RUN was performed with HT-1080 cells treated with (hIFN-γ) #8901 (50 ng/ml, 30 min) and Stat1 (D1K9Y) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human AIM2 promoter primers, human FZD5 promoter 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.
Simple Western™ analysis of lysates (0.1 mg/mL) from MCF-7 cells using Stat1 (D1K9Y) Rabbit mAb #14994. 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.
CUT&RUN was performed with HT-1080 cells and Stat1 (D1K9Y) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figure shows binding across FZD5 gene, a known target gene of Stat1 (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with HT-1080 cells and Stat1 (D1K9Y) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 2 (upper), including FZD5 gene (lower), a known target gene of Stat1 (see additional figure containing CUT&RUN-qPCR data).
Western blot analysis of extracts from HT-29 cells, untreated or treated with h-IFNa (10 ng/mL, 16 hr), followed by poly(I:C) (2.5 mg/mL, 7 hr) and MG-132 (#2194, 10mM, 7 hr) using Phospho-IRF-3 (Ser386) (E7J8G) XP® Rabbit mAb #37829 (upper), IRF-3 (D6I4C) XP® Rabbit mAb #11904 (middle), and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
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.
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).
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).
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.
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).
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).
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.
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.
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).
Western blot analysis of extracts from various cell lines, untreated (-) or UV-treated (2 hr recovery; +), using Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb.
Western blot analysis of extracts from various cell lines using IRF-3 (D6I4C) XP® Rabbit mAb.
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.
Western blot analysis of extracts from various cell lines using Stat1 (D1K9Y) Rabbit mAb.
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).
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.
Western blot analysis of extracts from various cell lines using IRF-9 (D2T8M) Rabbit mAb.
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).
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.
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.
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.
Immunohistochemical analysis of paraffin-embedded human ductal carcinoma of the breast using MX1 (D3W7I) Rabbit mAb performed on the Leica® Bond Rx.
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.
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).
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).
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Stat1 (D1K9Y) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded T84 (left, positive) and HCT116 (right, negative) cell pellets using MX1 (D3W7I) Rabbit mAb.
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).
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human lymphoma using Stat1 (D1K9Y) Rabbit mAb.
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.
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.
Immunohistochemical analysis of paraffin-embedded human lymphoma, control (left) or λ phosphatase-treated (right) using Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb.
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).
Immunohistochemical analysis of paraffin-embedded human non-small cell lung carcinoma using MX1 (D3W7I) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Phospho-Stat1 (Ser727) (D3B7) Rabbit mAb.
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.
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).
Immunohistochemical analysis of paraffin-embedded normal human spleen using MX1 (D3W7I) Rabbit mAb.
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.
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.
To Purchase # 43573
Cat. # Size Qty. Price
43573T
1 Kit  (9 x 20 microliters)

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
  • C&R
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).

Pathways

Explore pathways related to this product.

Limited Uses

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For Research Use Only. Not for Use in Diagnostic Procedures.
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