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NF-kappaB p65 Antibody Sampler Kit
NF-kappaB p65 Antibody Sampler Kit
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NF-kappaB p65 Antibody Sampler Kit

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    产品介绍
    产品信息
    抗原名称
    NF-kappaB p65
    来源纯化
    The NF-κB p65 (L8F6) Mouse mAb was produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human NF-κB p65. The Acetyl-NF-κB p65 (Lys310) (D2S3J) Rabbit mAb was produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Lys310 of human NF-κB p65 protein. The NF-κB p65 Antibody was produced by immunizing rabbits with a synthetic peptide corresponding to amino acids surrounding Glu498 of human NF-κB p65. The phospho-specific antibodies were produced by immunizing rabbits with synthetic phosphopeptides corresponding to amino acids surrounding the indicated target residues of human NF-κB p65. Polyclonal antibodies were purified by protein A and peptide affinity chromatography.
    简单描述
    Antibody Sampler Kit for studying Mouse IgG in the research area.
    应用
    目标/特异性

    Specificity/Sensitivity

    The total NF-κB p65 antibodies recognize endogenous levels of p65 regardless of post-translational modification state such as phosphorylation or acetylation. The phospho-NF-κB p65 antibodies recognize endogenous levels of p65 only when phosphorylated at their indicated target residues. The Acetyl-NF-κB p65 (Lys310) (D2S3J) Rabbit mAb recognizes transfected levels of p65 only when acetylated at Lys310.

    背景
    背景
    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.
    翻译后修饰
    Unmodified

    参考图片

    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.

    Immunohistochemical analysis of human chronic cholecystitis tissue using NF-κB p65 (L8F6) Mouse mAb.

    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.

    Flow cytometric analysis of HeLa cells, untreated (blue) or TNF-α-treated (green), using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb compared to a nonspecific negative control antibody (red).

    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).

    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).

    Confocal immunofluorescent analysis of HeLa cells, untreated (left) and 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).

    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).

    Western blot analysis of extracts from various cell lines using NF-κB p65 (L8F6) Mouse mAb #6956.Western免疫印迹。用NF-κB p65 (L8F6) Mouse mAb #6956研究各种细胞的细胞提取液。

    Western blot analysis of extracts from HeLa cells, mock transfected or transfected with NF-κB or NF-κB plus CBP, using Acetyl-NF-κB (Lys310) Antibody #3045. Western blots were competed with acetylated peptide (middle) or non-acetylated peptide (right).Western免疫印迹。用Acetyl-NF-κB (Lys310) Antibody #3045研究未转染的和转染了NF-κB 或 NF-κB plus CBP的HeLa细胞的细胞提取液。Western 免疫印迹与乙酰基化的多肽(中间图) 或未乙酰基化的多肽 (右图)比较。

    Western blot analysis of extracts from HeLa cells treated for 5 minutes with TNF-α #2169 (20 ng/ml), Calyculin A #9902 (50 nM), or both compounds, using Phospho-NF-kB p65 (Ser468) Antibody #3039 (top) or NF-kB p65 Antibody #3034 (bottom).Western免疫印迹。用Phospho-NF-kB p65 (Ser468) Antibody #3039 (上图) 或NF-kB p65 Antibody #3034 (下图)研究经TNF-α #2169 (20 ng/ml), Calyculin A #9902 (50 nM), 或者两种化合物处理5分钟的HeLa细胞的细胞提取液。

    Western blot analysis of extracts from various cell lines using NF-κB p65 (L8F6) Mouse mAb.

    Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (30 ng/ml, 1 hr) and either 10 μl of NF-κB p65 (L8F6) Mouse mAb or 2 μl of 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.

    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.

    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.

    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.

    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).

    Flow cytometric analysis of HeLa cells using NF-κB p65 (L8F6) Mouse mAb (blue) compared to a nonspecific negative control antibody (red).

    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).

    Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and either 5 μl of NF-κB p65 (D14E12) XP® Rabbit mAb or 2 μl of 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.

    Western blot analysis of extracts from various cell lines using NF-κB p65 (D14E12) XP® Rabbit mAb.

    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 #3033 (upper) or NF-κB p65 Antibody #3034 (lower).Western免疫印迹。用Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033 (上图) 或 NF-κB p65 Antibody #3034 (下图)研究未经处理的和经TNF-α (#2169, 20 ng/ml ,5 min) 处理的HeLa和NIH/3T3 细胞的细胞提取液。

    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).

    Immunohistochemical analysis of paraffin-embedded human chronic cholecystitis using NF-κB p65 (D14E12) XP® Rabbit mAb.

    Western blot analysis of extracts from various cell lines using NF-κB p65 (D14E12) XP® Rabbit mAb #8242.Western免疫印迹。用NF-κB p65 (D14E12) XP® Rabbit mAb #8242研究各种细胞的细胞提取液。

    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).

    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.

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    货号：
    4767T
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