Revision 1

#57103Store at -20C

1 Kit

(9 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
IRF-1 (D5E4) XP® Rabbit mAb 8478 20 µl 45-48 kDa Rabbit IgG
IRF-2 Antibody 4943 20 µl 45 kDa Rabbit 
IRF-3 (D6I4C) XP® Rabbit mAb 11904 20 µl 50-55 kDa Rabbit IgG
IRF-4 (D9P5H) Rabbit mAb 15106 20 µl 51 kDa Rabbit IgG
IRF-5 Antibody 20261 20 µl 60 kDa Rabbit 
IRF-6 Antibody 6948 20 µl 58, 60 kDa Rabbit 
IRF-7 (D2A1J) Rabbit mAb 13014 20 µl 65 kDa Rabbit IgG
IRF-8 (D20D8) Rabbit mAb 5628 20 µl 50 kDa Rabbit IgG
IRF-9 (D2T8M) Rabbit mAb 76684 20 µl 48 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The IRF Family Antibody Sampler Kit contains reagents to examine the total protein levels of various IRF isoforms. The kit contains enough primary and secondary antibodies to perform two western blot experiments with each primary antibody.

Storage

Polyclonal antibodies are supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol, and monoclonal antibodies also contain less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibodies.

Background

Interferon regulatory factors (IRFs) comprise a family of transcription factors that function within the Jak/Stat pathway to regulate interferon (IFN) and IFN-inducible gene expression in response to viral infection (1). IRFs play an important role in pathogen defense, autoimmunity, lymphocyte development, cell growth, and susceptibility to transformation. The IRF family includes nine members: IRF-1, IRF-2, IRF-9/ISGF3γ, IRF-3, IRF-4 (Pip/LSIRF/ICSAT), IRF-5, IRF-6, IRF-7, and IRF-8/ICSBP. All IRF proteins share homology in their amino-terminal DNA-binding domains. IRF family members regulate transcription through interactions with proteins that share similar DNA-binding motifs, such as IFN-stimulated response elements (ISRE), IFN consensus sequences (ICS), and IFN regulatory elements (IRF-E) (2).

  1. Taniguchi, T. et al. (2001) Annu Rev Immunol 19, 623-55.
  2. Honda, K. and Taniguchi, T. (2006) Nat Rev Immunol 6, 644-58.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
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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 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 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.

    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #57103

    IRF Family Antibody Sampler Kit

    IRF Family Antibody Sampler Kit: Image 1 Expand Image
    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.
    IRF Family Antibody Sampler Kit: Image 2 Expand Image
    Simple WesternTM analysis of lysates (1.0 mg/mL) from THP-1 cells using IRF-8 (D20D8) Rabbit mAb #5628. 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 JessTM Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    IRF Family Antibody Sampler Kit: Image 3 Expand Image
    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.
    IRF Family Antibody Sampler Kit: Image 4 Expand Image
    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).
    IRF Family Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from various cell lines using IRF-4 (D9P5H) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    IRF Family Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various cell lines using IRF-5 Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    IRF Family Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from SR, Ramos and A20 cells using IRF-2 Antibody.
    IRF Family Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from Raji and Jurkat cells, untreated or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml; overnight) using IRF-8 (D20D8) Rabbit mAb.
    IRF Family Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from various cell lines using IRF-6 Antibody. Absence of IRF-6 in MCF7 cells has been reported previously (5). Doublet has been reported to result from phosphorylation (6).
    IRF Family Antibody Sampler Kit: Image 10 Expand Image
    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.
    IRF Family Antibody Sampler Kit: Image 11 Expand Image
    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).
    IRF Family Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of HeLa cells, untreated or treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, 4 hr), using IRF-1 (D5E4) XP® Rabbit mAb.
    IRF Family Antibody Sampler Kit: Image 13 Expand Image
    CUT&RUN was performed with HeLa cells treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, 4 hr) and IRF-1 (D5E4) XP® 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 ERAP2, a known target gene of IRF-1 (see additional figure containing CUT&RUN-qPCR data).
    IRF Family Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of extracts from various cell lines using IRF-3 (D6I4C) XP® Rabbit mAb.
    IRF Family Antibody Sampler Kit: Image 15 Expand Image
    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.
    IRF Family Antibody Sampler Kit: Image 16 Expand Image
    Immunoprecipitation of IRF-4 from RPMI 8226 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is IRF-4 (D9P5H) Rabbit mAb. Western blot analysis was performed using IRF-4 (D9P5H) Rabbit mAb.
    IRF Family Antibody Sampler Kit: Image 17 Expand Image
    Immunoprecipitation of IRF-5 from HDLM-2 cell extracts. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is IRF-5 Antibody. Western blot analysis was performed using IRF-5 Antibody. Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) #93702 was used for detection.
    IRF Family Antibody Sampler Kit: Image 18 Expand Image
    Western blot analysis of extracts from THP-1, RL, and A20 cell lines using IRF-8 (D20D8) Rabbit mAb.
    IRF Family Antibody Sampler Kit: Image 19 Expand Image
    Western blot analysis of extracts from various cell lines using IRF-9 (D2T8M) Rabbit mAb.
    IRF Family Antibody Sampler Kit: Image 20 Expand Image
    Western blot analysis of extracts from various cell lines using IRF-1 (D5E4) XP® Rabbit mAb.
    IRF Family Antibody Sampler Kit: Image 21 Expand Image
    CUT&RUN was performed with HeLa cells treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, 4 hr) and IRF-1 (D5E4) XP® 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 figures show binding across chromosome 5 (upper), including ERAP2 (lower), a known target gene of IRF-1 (see additional figure containing CUT&RUN-qPCR data).
    IRF Family Antibody Sampler Kit: Image 22 Expand Image
    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.
    IRF Family Antibody Sampler Kit: Image 23 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from H929 cells and IRF-4 (D9P5H) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across SSR2 gene. For additional ChIP-seq tracks, please download the product data sheet.
    IRF Family Antibody Sampler Kit: Image 24 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected or transfected with human and mouse IRF-8 constructs, using IRF-8 (D20D8) Rabbit mAb.
    IRF Family Antibody Sampler Kit: Image 25 Expand Image
    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.
    IRF Family Antibody Sampler Kit: Image 26 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse thymus using IRF-1 (D5E4) XP® Rabbit mAb performed on the Leica® BOND™ Rx.
    IRF Family Antibody Sampler Kit: Image 27 Expand Image
    CUT&RUN was performed with HeLa cells treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, 4 hr) and either IRF-1 (D5E4) XP® 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 MOV10 intron 1 primers, human ERAP2 intron 1, 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.
    IRF Family Antibody Sampler Kit: Image 28 Expand Image
    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).
    IRF Family Antibody Sampler Kit: Image 29 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from H929 cells and IRF-4 (D9P5H) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 1 (upper), including SSR2 (lower).
    IRF Family Antibody Sampler Kit: Image 30 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from THP-1 and IRF-8 (D20D8) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across GPR18/UBAC2, a known target gene of IRF8 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.
    IRF Family Antibody Sampler Kit: Image 31 Expand Image
    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).
    IRF Family Antibody Sampler Kit: Image 32 Expand Image
    Imunohistochemical analysis of paraffin-embedded human breast carcinoma using IRF-1 (D5E4) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
    IRF Family Antibody Sampler Kit: Image 33 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from H929 cells and either IRF-4 (D9P5H) 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 PRDM1 Exon 2 primers, SimpleChIP® Human SUB1 Promoter Primers #5156 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.
    IRF Family Antibody Sampler Kit: Image 34 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from THP-1 and IRF-8 (D20D8) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 13 (upper), including GPR18/UBAC2 (lower), a known target gene of IRF8 (see additional figure containing ChIP-qPCR data).
    IRF Family Antibody Sampler Kit: Image 35 Expand Image
    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.
    IRF Family Antibody Sampler Kit: Image 36 Expand Image
    Imunohistochemical analysis of paraffin-embedded human lymphoma using IRF-1 (D5E4) XP® Rabbit mAb.
    IRF Family Antibody Sampler Kit: Image 37 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 THP-1 and either IRF-8 (D20D8) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using human GPR18 promoter primers, SimpleChIP® Human MS4A7 Promoter Primers #9013, 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.
    IRF Family Antibody Sampler Kit: Image 38 Expand Image
    Immunohistochemical analysis of paraffin-embedded mosue thymus using IRF-1 (D5E4) XP® Rabbit mAb.
    IRF Family Antibody Sampler Kit: Image 39 Expand Image
    Imunohistochemical analysis of paraffin-embedded HeLa cell pellets, control (left) or IFN gamma-treated (right), using IRF-1 (D5E4) XP® Rabbit mAb.
    IRF Family Antibody Sampler Kit: Image 40 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, serum-starved (left) or treated with hIFNγ #8901 (100 ng/mL, 4 hr; right), using IRF-1 (D5E4) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin.
    IRF Family Antibody Sampler Kit: Image 41 Expand Image
    Flow cytometric analysis of HeLa cells, untreated (blue) or treated with Human Interferon-γ (hIFN-γ) #8901 (green), using IRF-1 (D5E4) XP® Rabbit mAb.
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.