Revision 1

#4767Store at -20C

1 Kit

(5 x 20 microliters)

Cell Signaling Technology

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

Support: 877-678-TECH (8324)

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb 3033 20 µl 65 kDa Rabbit IgG
Acetyl-NF-κB p65 (Lys310) (D2S3J) Rabbit mAb 12629 20 µl 65 kDa Rabbit IgG
NF-κB p65 (L8F6) Mouse mAb 6956 20 µl 65 kDa Mouse IgG2b
NF-κB p65 (D14E12) XP® Rabbit mAb 8242 20 µl 65 kDa Rabbit IgG
Phospho-NF-κB p65 (Ser468) Antibody 3039 20 µl 65 kDa Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl Horse 

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

Description

The NF-κB p65 Antibody Sampler Kit contains reagents to examine NF-κB p65/RelA phosphorylation at Ser468 and Ser536; acetylation at Lys310; and total p65 levels.

Storage

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

Background

Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammatory and immune responses (1,2). There are five family members in mammals: RelA, c-Rel, RelB, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Both p105 and p100 are proteolytically processed by the proteasome to produce p50 and p52, respectively. Rel proteins bind p50 and p52 to form dimeric complexes that bind DNA and regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins (3-5). NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression (6-8). NIK and IKKα (IKK1) regulate the phosphorylation and processing of NF-κB2 (p100) to produce p52, which translocates to the nucleus (9-11).
RelA/p65 is a subunit of the NF-κB transcription complex, which plays a crucial role in inflammatory and immune responses. The complex is composed of various homodimeric and heterodimeric Rel family member combinations, the activity of which is modulated by post-translational modifications including phosphorylation and acetylation. p65 phosphorylation by PKA and/or MSK1 at Ser276 allows for increased interaction with the transcriptional coactivator p300/CBP to enhance transcriptional activity. NF-κB dimer assembly with IκB, as well as its DNA binding and transcriptional activities, are regulated by p300/CBP acetyltransferases that principally target Lys218, Lys221 and Lys310 (12-14). This process is reciprocally regulated by histone deacetylases (HDACs); several HDAC inhibitors have been shown to activate NF-κB (12-14). T-cell co-stimulation and Calyculin A have both been shown to increase Ser468 phosphorylation (15,16). IKKβ (but not IKKα) and GSK-3β both target this site (16,17), which appears to have a negative regulatory role not involving inhibition of nuclear translocation after TNF-α or IL-1β stimulation (17). p65 phosphorylation at Ser536 regulates activation, nuclear localization, protein-protein interactions, transcriptional activity, and apoptosis (18-22).

  1. Baeuerle, P.A. and Henkel, T. (1994) Annu Rev Immunol 12, 141-79.
  2. Baeuerle, P.A. and Baltimore, D. (1996) Cell 87, 13-20.
  3. Haskill, S. et al. (1991) Cell 65, 1281-9.
  4. Thompson, J.E. et al. (1995) Cell 80, 573-82.
  5. Whiteside, S.T. et al. (1997) EMBO J 16, 1413-26.
  6. Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.
  7. Scherer, D.C. et al. (1995) Proc Natl Acad Sci USA 92, 11259-63.
  8. Chen, Z.J. et al. (1996) Cell 84, 853-62.
  9. Senftleben, U. et al. (2001) Science 293, 1495-9.
  10. Coope, H.J. et al. (2002) EMBO J 21, 5375-85.
  11. Xiao, G. et al. (2001) Mol Cell 7, 401-9.
  12. Ashburner, B.P. et al. (2001) Mol Cell Biol 21, 7065-77.
  13. Mayo, M.W. et al. (2003) J Biol Chem 278, 18980-9.
  14. Chen, L.F. et al. (2002) EMBO J 21, 6539-48.
  15. Mattioli, I. et al. (2004) Blood 104, 3302-4.
  16. Buss, H. et al. (2004) J Biol Chem 279, 49571-4.
  17. Schwabe, R.F. and Sakurai, H. (2005) FASEB J 19, 1758-60.
  18. Doyle, S.L. et al. (2005) J Biol Chem 280, 23496-501.
  19. Sasaki, C.Y. et al. (2005) J Biol Chem 280, 34538-47.
  20. Mattioli, I. et al. (2004) J Immunol 172, 6336-44.
  21. Bae, J.S. et al. (2003) Biochem Biophys Res Commun 305, 1094-8.
  22. Buss, H. et al. (2004) J Biol Chem 279, 55633-43.

Background References

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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.
    Revision 1
    #4767

    NF-κB p65 Antibody Sampler Kit

    NF-κB p65 Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (1.0 mg/mL) from HeLa cells treated with hTNF-α (20 ng/mL, 5 minutes) using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033. The virtual lane view (left) shows a single 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.
    NF-κB p65 Antibody Sampler Kit: Image 2 Expand Image
    Immunoprecipitation of Phospho-NF-κB p65 (Ser536) from HeLa extracts treated with hTNF-α #8902 (20 ng/ml, 5 min). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb. Western blot analysis was performed using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
    NF-κB p65 Antibody Sampler Kit: Image 3 Expand Image
    Immunoprecipitation of NF-kB p65 from HeLa cell extracts. Lane 1 is 10% input, lane 2 is precipitated with Mouse (G3A1) mAb IgG1 Isotype Control #5415, and lane 3 is NF-κB p65 (L8F6) Mouse mAb, #6956. Western blot was performed using NF-κB p65 (D14E12) XP® Rabbit mAb, #8242.
    NF-κB p65 Antibody Sampler Kit: Image 4 Expand Image

    Immunoprecipitation of NF-kB p65 from HeLa cell extracts. Lane 1 is 10% input, lane 2 is precipitated with Mouse (G3A1) mAb IgG1 Isotype Control #5415, and lane 3 is NF-κB p65 (L8F6) Mouse mAb, #6956. Western blot was performed using NF-κB p65 (D14E12) XP® Rabbit mAb, #8242.

    NF-κB p65 Antibody Sampler Kit: Image 5 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from HeLa cells using NF-κB p65 (D14E12) XP® Rabbit mAb #8242. The virtual lane view (left) shows a single 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.
    NF-κB p65 Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged human NF-κB p65 (hNF-κB p65; +) and HA-tagged human p300 (hp300-HA; +), using Acetyl-NF-κB p65 (Lys310) (D2S3J) Rabbit mAb (upper) or NF-κB p65 (D14E12) XP® Rabbit mAb #8242 (lower).
    NF-κB p65 Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated or TNF-α treated (#2169, 20 ng/ml for 5 minutes), using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (upper) or NF-κB p65 Antibody #3034 (lower).
    NF-κB p65 Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from HeLa cells treated for 5 minutes with TNF-alpha #2169 (20 ng/ml), Calyculin A #9902 (50 nM), or both compounds, using Phospho-NF-kappaB p65 (Ser468) Antibody (top) or NF-kappaB p65 Antibody #3034 (bottom).
    NF-κB p65 Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from control HeLa cells (lane 1) or NF-κB p65 knockout HeLa cells (lane 2) using NF-κB p65 (L8F6) Mouse mAb #6956 (upper) or β-actin (13E5) Rabbit mAb #4970 (lower). The absence of signal in the NF-κB p65 knockout HeLa cells confirms the specificity of the antibody for NF-κB p65.
    NF-κB p65 Antibody Sampler Kit: Image 10 Expand Image
    Flow cytometric analysis of MCF7 cells using NF-kB p65 (L8F6) Mouse mAb (solid line) or concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
    NF-κB p65 Antibody Sampler Kit: Image 11 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.
    NF-κB p65 Antibody Sampler Kit: Image 12 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.
    NF-κB p65 Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from various cell lines using NF-κB p65 (D14E12) XP® Rabbit mAb.
    NF-κB p65 Antibody Sampler Kit: Image 14 Expand Image
    Immunoprecipitation of acetyl-NF-κB p65 (Lys310) from 293T cells, cotransfected with Myc/DDK-tagged human NF-κB p65 and HA-tagged human p300, using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Acetyl-NF-κB p65 (Lys310) (D2S3J) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Acetyl-NF-κB p65 (Lys310) (D2S3J) Rabbit mAb.
    NF-κB p65 Antibody Sampler Kit: Image 15 Expand Image
    Western blot analysis of extracts from THP-1 cells, differentiated with TPA (#9905, 80 nM for 24h) and treated with 1 μg/ml LPS for the indicated times, using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (upper) and NF-κB p65 (C22B4) Rabbit mAb #4764 (lower).
    NF-κB p65 Antibody Sampler Kit: Image 16 Expand Image
    Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® NF-κB p65 siRNA I #6261 (+), using NF-κB p65 (L8F6) Mouse mAb (upper) or α-Tubulin (11Η10) Rabbit mAb #2125 (lower). The NF-κB p65 (L8F6) Mouse mAb confirms silencing of NF-κB p65 expression, while the α-Tubulin (11Η10) Rabbit mAb is used as a loading control.
    NF-κB p65 Antibody Sampler Kit: Image 17 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, serum starved (left) or TNF-α treated (#8902 at 20 ng/ml for 20 min, right), using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® phalloidin 555 (red).
    NF-κB p65 Antibody Sampler Kit: Image 18 Expand Image
    Western blot analysis of extracts from various cell lines using NF-κB p65 (L8F6) Mouse mAb.
    NF-κB p65 Antibody Sampler Kit: Image 19 Expand Image
    Immunohistochemical analysis using NF-κB p65 (D14E12) XP® Rabbit mAb on SignalSlide® NF-κB p65 IHC Controls #12873 (paraffin-embedded HCT116 cells, untreated (left) or treated with hTNF-α #8902 (right)).
    NF-κB p65 Antibody Sampler Kit: Image 20 Expand Image
    Flow cytometric analysis of HeLa cells, untreated (blue) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 and Calyculin A #9902 (20 ng/ml and 100 nM, 15 min; green), using Phospho-NF-κB p65 (Ser536) (93H1) 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.
    NF-κB p65 Antibody Sampler Kit: Image 21 Expand Image
    Immunohistochemical analysis of human chronic cholecystitis tissue using NF-κB p65 (L8F6) Mouse mAb.
    NF-κB p65 Antibody Sampler Kit: Image 22 Expand Image
    Immunohistochemical analysis of paraffin-embedded human chronic cholecystitis using NF-κB p65 (D14E12) XP® Rabbit mAb.
    NF-κB p65 Antibody Sampler Kit: Image 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded OVCAR8 cell pellets treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (left) or treated with SignalSilence® NF-κB p65 siRNA I #6261 (right), using NF-κB p65 (L8F6) Mouse mAb.
    NF-κB p65 Antibody Sampler Kit: Image 24 Expand Image
    Confocal immunofluorescent analysis of HT-1080 cells, untreated (left) or treated with hTNF-α #8902 (20 ng/ml, 20 min) (right), using NF-κB p65 (D14E12) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    NF-κB p65 Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, untreated (left) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (right), using NF-κB p65 (L8F6) Mouse mAb.
    NF-κB p65 Antibody Sampler Kit: Image 26 Expand Image
    Flow cytometric analysis of HeLa cells using NF-κB p65 (D14E12) XP® Rabbit mAb (blue) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red).
    NF-κB p65 Antibody Sampler Kit: Image 27 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (20 ng/mL, 20 min; right), using NF-κB p65 (L8F6) Mouse mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    NF-κB p65 Antibody Sampler Kit: Image 28 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using SimpleChIP® 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 IL-8, a known target gene of NFκB (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product datasheet.
    NF-κB p65 Antibody Sampler Kit: Image 29 Expand Image
    Flow cytometric analysis of MCF7 cells using NF-kB p65 (L8F6) Mouse mAb (solid line) compared to a concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
    NF-κB p65 Antibody Sampler Kit: Image 30 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using SimpleChIP® 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 4 (upper), including IL-8 (lower), a known target gene of NFκB (see additional figure containing ChIP-qPCR data).
    NF-κB p65 Antibody Sampler Kit: Image 31 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (30 ng/ml, 1 hr) and either NF-κB p65 (L8F6) Mouse 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 IκBα Promoter Primers #5552, human IL-8 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.
    NF-κB p65 Antibody Sampler Kit: Image 32 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and either NF-κB p65 (D14E12) XP® 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 IκBα Promoter Primers #5552, human IL-8 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.
    NF-κB p65 Antibody Sampler Kit: Image 33 Expand Image
    CUT&RUN was performed with HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® 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 figure shows binding across LAMC2, a known target gene of NF-κB p65 (see additional figure containing CUT&RUN-qPCR data).
    NF-κB p65 Antibody Sampler Kit: Image 34 Expand Image
    CUT&RUN was performed with HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® 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 1 (upper), including LAMC2 (lower), a known target gene of NF-κB p65 (see additional figure containing CUT&RUN-qPCR data).
    NF-κB p65 Antibody Sampler Kit: Image 35 Expand Image
    CUT&RUN was performed with HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and either NF-κB p65 (D14E12) 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 LAMC2 upstream primers, and human ITM2A upstream primers. 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.
    NF-κB p65 Antibody Sampler Kit: Image 36 Expand Image
    Immunoprecipitation of NF-kB p65 from CHO cell extracts. Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is NF-κB p65 (D14E12) XP® Rabbit mAb, #8242. Western blot was performed using NF-κB p65 (L8F6) Mouse mAb, #6956.
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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.