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35114
IFN (Type I/III) Signaling Pathway Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit #35114

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IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 1

Western blot analysis of extracts from various cell lines using Tyk2 (D4I5T) Rabbit mAb.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 2

Western blot analysis of extracts from serum-starved U266 and Jurkat cells, untreated (-) or treated with Human Interferon-α1 (hIFN-α1) #8927 (50 ng/ml, 15 min; +) using Phospho-Tyk2 (Tyr1054/1055) (D7T8A) Rabbit mAb (upper) or total Tyk2 (D4I5T) Rabbit mAb #14193 (lower).

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 3

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Jak1 (6G4) Rabbit mAb #3344, in the presence of control peptide (left) or Jak1 Blocking Peptide (right).

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 4

Western blot analysis of extracts from various cell lines, serum-starved overnight (-) followed by treatment with Human Interferon-α1 #8927 (hIFN-α1, 10 ng/ml, 15 min; +) or Human Interleukin-4 #8919 (hIL-4, 10 ng/ml, 10 min; +) using Phospho-Jak1 (Tyr1034/1035) (D7N4Z) Rabbit mAb (upper) or Jak1 (6G4) Rabbit mAb (lower).

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 5

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.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 6

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.

IFN (Type I/III) Signaling Pathway 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 Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across IRF-1, a known target gene of Phospho-Stat1 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 8

Chromatin immunoprecipitations were performed with cross-linked chromatin from U266 cells treated with Human Interferon-α (IFN-α) #9906 (10nM) for 30 min and Stat2 (D9J7L) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across USP18, a known target gene of Stat2 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 9

Flow cytometric analysis of U266 cells, untreated (blue) or treated with IFN-α (green) using Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor 488 Conjugate) #4412 was used as a secondary antibody.

IFN (Type I/III) Signaling Pathway 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.

IFN (Type I/III) Signaling Pathway 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).

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: 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.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 13

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length human Tyk2 (hTyk2; +), using Tyk2 (D4I5T) Rabbit mAb.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 14

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Jak1 (6G4) Rabbit mAb.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 15

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.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 16

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HT-1080 cells treated with IFN-γ (50 ng/ml) for 30 minutes and 5 μl of Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 5 (upper), including IRF1 (lower), a known target gene of Phospho-Stat1 (see additional figure containing ChIP-qPCR data).

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 17

Chromatin immunoprecipitations were performed with cross-linked chromatin from U266 cells treated with Human Interferon-α (IFN-α) #9906 (10nM) for 30 min and Stat2 (D9J7L) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 22 (upper), including USP18 (lower), a known target gene of Stat2 (see additional figure containing ChIP-qPCR data).

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 18

Confocal immunofluorescent analysis of A-431 cells, serum starved (left) or treated with IFNα (1000 U/ml for 30 min; right) using Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb (green) and β-actin (8H10D10) Mouse mAb #3700 (red).

IFN (Type I/III) Signaling Pathway 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).

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 20

Western blot analysis of extracts from MCF7 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Tyk2 siRNA #14254 (+), using Tyk2 (D4I5T) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The Tyk2 (D4I5T) Rabbit mAb confirms silencing of Tyk2 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 21

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Jak1 (6G4) Rabbit mAb.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 22

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).

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 23

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 (Tyr701) (D4A7) 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.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 24

Chromatin immunoprecipitations were performed with cross-linked chromatin from U266 cells treated with Human Interferon-α (IFN-α) #9906 (100 ng/ml) for 30 min, and either Stat2 (D9J7L) 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 USP18 promoter primers, SimpleChIP® Human WARS Intron 1 Primers #30101, 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.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 25

Western blot analysis of extracts from serum-starved U266 and A-431 cells, untreated (-) or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml; +) using Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb (upper) and total Stat2 (D9J7L) Rabbit mAb #72604 (lower).

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 26

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.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 27

Western blot analysis of extracts from ACHN, SR, CTLL-2 and PC12 cell lines using Jak1 (6G4) Rabbit mAb.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 28

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

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 29

Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with hIFN-α1 #8927 (green), using Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 30

Flow cytometric analysis of U266 cells using Stat2 (D9J7L) 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 647 Conjugate) was used as a secondary antibody.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 31

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

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 32

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

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 33

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with hIFN-α1 #8927 (100 ng/mL, 30 min; right), using Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb (green) and β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116 (red).

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 34

Confocal immunofluorescent analysis of A-431 cells, serum starved (left) or treated with IFNα (1000 U/ml for 30 mins; right) using Stat2 (D9J7L) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

IFN (Type I/III) Signaling Pathway 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.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 36

Western blot analysis of extracts from HeLa, A20, and PC-12 cells, untreated or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml, 30 min), using Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb (upper) or Stat1 Antibody #9172 (lower).

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 37

Immunoprecipitation of Stat2 from KARPAS-299 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Stat2 (D9J7L) Rabbit mAb. Western blot was performed using Stat2 (D9J7L) Rabbit mAb. KARPAS cell line source: Dr Abraham Karpas at the University of Cambridge.

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 38

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

IFN (Type I/III) Signaling Pathway Antibody Sampler Kit: Image 39

Western blot analysis of extracts from various cell lines using Stat2 (D9J7L) Rabbit mAb. KARPAS cell line source: Dr Abraham Karpas at the University of Cambridge.

To Purchase # 35114T
Product # Size Price
35114T
1 Kit  (9 x 20 µl) $ 626

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Tyk2 (D4I5T) Rabbit mAb 14193 20 µl
  • WB
  • IP
H Mk 134 Rabbit IgG
Phospho-Tyk2 (Tyr1054/1055) (D7T8A) Rabbit mAb 68790 20 µl
  • WB
H 134 Rabbit IgG
Jak1 (6G4) Rabbit mAb 3344 20 µl
  • WB
  • IHC
H M R 130 Rabbit IgG
Phospho-Jak1(Tyr1034/1035) (D7N4Z) Rabbit mAb 74129 20 µl
  • WB
H M 130 Rabbit IgG
Stat1 (D1K9Y) Rabbit mAb 14994 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H M R Mk 84, 91 Rabbit IgG
Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb 7649 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M R 84, 91 Rabbit IgG
Stat2 (D9J7L) Rabbit mAb 72604 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M 97, 113 Rabbit IgG
Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb 88410 20 µl
  • WB
  • IF
  • F
H R 97, 113 Rabbit IgG
IRF-9 (D2T8M) Rabbit mAb 76684 20 µl
  • WB
  • IP
  • IF
  • ChIP
H 48 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The IFN (Type I/III) Signaling Pathway Antibody Sampler Kit provides an economical means of detecting the activation of the IFN (Type I/III) signaling pathway using phospho-specific and control antibodies. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Each antibody in the IFN (Type I/III) Signaling Pathway Antibody Sampler kit detects endogenous levels of its target protein. Phospho-Jak1 (Tyr1034/1035) (D7N4Z) Rabbit mAb can detect Jak1 when dually or singly phosphorylated at Tyr1034. This site has historically been referenced as Tyr1022 and Ty1023. Phospho-Tyk2 (Tyr1054/1055) (D7T8A) Rabbit mAb detects Tyk2 when phosphorylated at both Tyr1054 and Tyr1055. Cross-reactivity was not observed with other family members. Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb recognizes endogenous levels of Stat1 protein only when phosphorylated at Tyr701. Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb recognizes endogenous levels of Stat2 protein only when phosphorylated at Tyr690. IRF-9 (D2T8M) Rabbit mAb cross-reacts with an unidentified protein of 95 kDa. Stat1 (D1K9Y) Rabbit mAb cross-reacts with an unidentified protein of 150 kDa. A band of unknown identity at 55 kDa is detected in some cell lines by Tyk2 (D4I5T) Rabbit mAb.

Source / Purification

Monoclonal antibodies are produced by immunizing rabbits with synthetic peptides corresponding to residues surrounding Ile800 of Jak1, Pro688 of Stat1, Leu706 of Stat2, the carboxy terminus of Tyk2, and with recombinant protein specific to IRF-9. Phosphorylation-specific monoclonal antibodies are produced by immunizing rabbits with synthetic peptides corresponding to residues surrounding Tyr1034/1035 of Jak1, Tyr1054/1055 of Tyk2, Tyr701 of Stat1, and Tyr690 of Stat2.

Background

Originally discovered in the late 1950s for their antiviral activity, interferons (IFNs) have since been assigned diverse roles in many physiological and pathological processes. There are three families of IFNs: types I, II, and III. In humans, type I contains IFN-α (13 different subtypes), IFN-β (also known as IFN-β1), IFN-ε, IFN-κ, and IFN-ω. They bind to a receptor complex containing IFNAR1 and IFNAR2, which is broadly expressed on most cells. IFN-γ is the sole member of type II IFN. It signals through a receptor complex consisting of IFNγR1 and IFNγR2, which is also expressed on most cell types. Type III IFN, also known as interferon lambdas (IFN-λs), have four members in humans: IFN-λ1 (IL29), IFN-λ2 (IL28A), IFN-λ3 (IL28B), and IFN-λ4. IFN-λs signal through a heterodimeric receptor comprised of IFNλR1 and IL-10R2. While IL-10R2 is broadly expressed and shared by the IL-10 family cytokines, IFNλR1 expression is restricted to epithelial cells, neuronal cells, and subsets of myeloid cells (1-3). Engagement of all IFNs with their receptors initiates downstream signaling events, mainly, activation of the Jak–Stat signaling cascade. For type I and III IFNs, Jak1 and Tyk2 are phosphorylated and activated, leading to subsequent phosphorylation of Stat1 and Stat2. Phosphorylated Stat1 and Stat2 are released from the receptor complex and, together with IRF-9, they form so-called ISGF3 (interferon-stimulated gene factor 3) transcriptional complex. ISGF3 translocates to the nucleus, binds to the interferon-stimulated response element (ISRE) to initiate the transcription of a wide array of interferon-stimulated genes (ISGs) (4,5). On the other hand, IFN-γ induces phosphorylation and activation of Jak1 and Jak2, which subsequently phosphorylate Stat1. Phosphorylated Stat1 dimerizes, translocates to the nucleus, and binds to γ-interferon-activated site (GAS) to initiate the transcription of ISGs (6,7).

  1. Schneider, W.M. et al. (2014) Annu Rev Immunol 32, 513-45.
  2. Hemann, E.A. et al. (2017) Front Immunol 8, 1707.
  3. Walter, M.R. (2020) Front Immunol 11, 606489.
  4. Hervas-Stubbs, S. et al. (2011) Clin Cancer Res 17, 2619-27.
  5. Mesev, E.V. et al. (2019) Nat Microbiol 4, 914-924.
  6. Green, D.S. et al. (2017) J Biol Chem 292, 13925-13933.
  7. Ivashkiv, L.B. (2018) Nat Rev Immunol 18, 545-558.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

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.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Jak antibodies produced under license (granting certain rights including those under U.S. Patent No. 5,658,791) from Chemicon International, Inc.