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44902
IFN-γ Signaling Pathway Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

IFN-γ Signaling Pathway Antibody Sampler Kit #44902

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IFN-γ Signaling Pathway Antibody Sampler Kit: Image 1

Immunoprecipitation of IFNGR1 from NCI-H226 cell extracts. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is IFNGR1 (E444) Antibody. Western blot analysis was performed using IFNGR1 (E444) Antibody. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used for detection.

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 2

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-γ Signaling Pathway Antibody Sampler Kit: Image 3

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-γ Signaling Pathway Antibody Sampler Kit: Image 4

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Jak2 (D2E12) XP® Rabbit mAb.

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 5

Western blot analysis of extracts from UT-7 and BaF3 cells, untreated or treated with GM-CSF or IL-3 (5 minutes), using Phospho-Jak2 (Tyr1007/1008) (C80C3) Rabbit mAb #3776 (top) or total Jak2 (D2E12) XP® Rabbit mAb #3230 (bottom).

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 6

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-γ 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 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.

IFN-γ Signaling Pathway 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.

IFN-γ Signaling Pathway 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.

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 10

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-γ Signaling Pathway Antibody Sampler Kit: Image 11

Western blot analysis of extracts from various cell lines with IFNGR1 (E444) Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 12

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

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 13

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Jak2 (D2E12) XP® Rabbit mA in the presence of control peptide (left) or Jak2 Blocking Peptide #1039 (right).

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 14

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-γ Signaling Pathway Antibody Sampler Kit: Image 15

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

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 16

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-γ Signaling Pathway Antibody Sampler Kit: Image 17

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human IFNGR1 protein (hIFNGR1-Myc/DDK; +), using IFNGR1 (E444) Antibody (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 18

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

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 19

Western blot analysis of extracts from K-562 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or Jak2 siRNA (+), using Jak2 (D2E12) XP® Rabbit mAb #3230 and α-Tubulin (11H10) Rabbit mAb #2125. The Jak2 (D2E12) XP® Rabbit mAb confirms silencing of Jak2 expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of Jak2 siRNA.

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 20

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-γ Signaling Pathway Antibody Sampler Kit: Image 21

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

IFN-γ 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-γ Signaling Pathway Antibody Sampler Kit: Image 23

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

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 24

Western blot analysis of extracts from K-562, THP-1, TF-1 and BaF3 cell lines using Jak2 (D2E12) XP® Rabbit mAb.

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 25

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

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 26

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

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 27

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

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 28

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-γ Signaling Pathway Antibody Sampler Kit: Image 29

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

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 30

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

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 31

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-γ Signaling Pathway Antibody Sampler Kit: Image 32

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).

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 33

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-γ Signaling Pathway Antibody Sampler Kit: Image 34

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).

IFN-γ Signaling Pathway Antibody Sampler Kit: Image 35

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

To Purchase # 44902T
Product # Size Price
44902T
1 Kit  (9 x 20 µl) $ 626

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
IFNGR1 (E444) Antibody 10405 20 µl
  • WB
  • IP
H 45-90 Rabbit 
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
Jak2 (D2E12) XP® Rabbit mAb 3230 20 µl
  • WB
  • IP
  • IHC
H M R 125 Rabbit IgG
Phospho-Jak2 (Tyr1007/1008) (C80C3) Rabbit mAb 3776 20 µl
  • WB
H M R 125 Rabbit IgG
Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb 7649 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M R 84, 91 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
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The IFN-γ Signaling Pathway Antibody Sampler Kit provides an economical means of detecting the activation of the IFN-γ 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-γ 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-Jak2 (Tyr1007/1008) (C80C3) Rabbit mAb detects endogenous levels of Jak2 when phosphorylated at Tyr1007/1008. This antibody can also detect single phosphorylation at either 1007 or 1008. This antibody may cross-react with phospho-Jak1 and phospho-Tyk2. Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb recognizes endogenous levels of Stat1 protein only when phosphorylated at Tyr701. 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.

Source / Purification

Monoclonal antibodies are produced by immunizing rabbits with synthetic peptides corresponding to residues surrounding Ile800 of Jak1, Pro841 of Jak2, and Pro688 of Stat1. Phosphorylation-specific monoclonal antibodies are produced by immunizing rabbits with synthetic peptides corresponding to residues surrounding Tyr1034/1035 of Jak1, Tyr1007/10008 of Jak2, and Tyr701 and Ser727 of Stat1. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Glu444 of IFNGR1 protein. Antibodies are purified by protein A and peptide affinity chromatography.

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.
XP is a registered 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.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.