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68309
Notch Activated Targets Antibody Sampler Kit

Notch Activated Targets Antibody Sampler Kit #68309

Western Blotting Image 1

Western blot analysis of extracts from various cell lines using HES1 (D6P2U) Rabbit mAb #11988.

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Western Blotting Image 2

Western blot analysis of extracts from various cell lines using MAML1 (D3K7B) Rabbit mAb #12166.

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Western Blotting Image 3

Western blot analysis of extracts from various cell lines using c-Myc (D3N8F) Rabbit mAb #13987.

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Western Blotting Image 4

Western blot analysis of extracts from SK-N-MC, C6 and IMCD3 cells, using Cyclin D3 (DCS22) Mouse mAb #2936.

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Western Blotting Image 5

Western blot analysis of extracts from various cell types using p21 Waf1/Cip1 (12D1) Rabbit mAb #2947.

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Western Blotting Image 6

Western blot analysis of total cell extract from various cell types using Notch1 (D1E11) XP® Rabbit mAb #3608. The full-length (FL) Notch protein and the cleaved transmembrane/intracellular region (NTM) are indicated.

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Western Blotting Image 7

Western blot analysis of extracts from various cell lines using Cleaved Notch1 (Val1744) (D3B8) Rabbit mAb #4147 (upper) or Notch1 (D1E11) XP® Rabbit mAb #3608 (lower).

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Western Blotting Image 8

Western blot analysis of extracts from various cell lines using RBPSUH (D10A4) XP® Rabbit mAb #5313. (D10A4) XP® Rabbit mAb #5313.

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Western Blotting Image 9

Western blot analysis of total cell extract from various cell types using Notch1 (D1E11) XP® Rabbit mAb. The full-length (FL) Notch protein and the cleaved transmembrane/intracellular region (NTM) are indicated.

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Western Blotting Image 10

Western blot analysis of extracts from various cell lines using Cleaved Notch1 (Val1744) (D3B8) Rabbit mAb (upper) or Notch1 (D1E11) XP® Rabbit mAb #3608 (lower).

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Chromatin IP Image 11

CUTLL1 cells were cultured in media with γ-secretase inhibitor (1 μM, 3 d) and then either harvested immediately (left panel) or washed and cultured in fresh media for 3 h (right panel). Chromatin immunoprecipitations were performed with cross-linked chromatin from cells and RBPSUH (D10A4) 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 human HES1 promoter primers, SimpleChIP® Human HES4 Promoter Primers #7273, 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.

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Western Blotting Image 12

Western blot analysis of extracts from various cell lines using RBPSUH (D10A4) XP® Rabbit mAb.

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Western Blotting Image 13

Western blot analysis of extracts from various cell lines using MAML1 (D3K7B) Rabbit mAb.

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Chromatin IP-seq Image 14

Chromatin immunoprecipitations were performed with cross-linked chromatin from Daudi cells and c-Myc (D3N8F) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared from 5 ng enriched ChIP DNA using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, and sequenced on the Illumina NextSeq. The figure shows binding across NPM1, a known target gene of c-Myc (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

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Western Blotting Image 15

Western blot analysis of extracts from various cell lines using c-Myc (D3N8F) Rabbit mAb.

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Western Blotting Image 16

Western blot analysis of extracts from various cell types using p21 Waf1/Cip1 (12D1) Rabbit mAb.

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Western Blotting Image 17

Western blot analysis of extracts from various cell lines using HES1 (D6P2U) Rabbit mAb.

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IHC-P (paraffin) Image 18

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using HES1 (D6P2U) Rabbit mAb.

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Western Blotting Image 19

Western blot analysis of extracts from SK-N-MC, C6 and IMCD3 cells, using Cyclin D3 (DCS22) Mouse mAb.

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Western Blotting Image 20

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.

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IHC-P (paraffin) Image 21

Immunohistochemical analysis of paraffin-embedded human inflammatory granulation tissue using Notch1 (D1E11) XP® Rabbit mAb.

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Chromatin IP Image 22

CUTLL1 cells were cultured in media with γ-secretase inhibitor (1μM) for 3 days and then either harvested immediately (left panel) or washed and cultured in fresh media for 3h (right panel). Chromatin immunoprecipitations were performed with cross-linked chromatin from cells and Cleaved Notch1 (Val1744) (D3B8) 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 HES1 promoter primers, SimpleChIP® Human HES4 Promoter Primers #7273, 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.

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IHC-P (paraffin) Image 23

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using RBPSUH (D10A4) XP® Rabbit mAb.

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IP Image 24

Immunoprecipitation of MAML1 protein from 293T cell extracts, using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or MAML1 (D3K7B) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using MAML1 Antibody #4608.

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Chromatin IP Image 25

Chromatin immunoprecipitations were performed with cross-linked chromatin from Daudi cells and either c-Myc (D3N8F) 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 ATF4 promoter primers, SimpleChIP® Human NPM1 Intron 1 Primers #4779, 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.

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Western Blotting Image 26

Western blot analysis of extracts from HT-29 cells, untreated (-) or treated with JQ1 (1 μM; 72 hr; +), using c-Myc (D3N8F) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, the BET bromodomain inhibitor JQ1 inhibits c-Myc expression.

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Western Blotting Image 27

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® p21 Waf1/Cip1 siRNA II (+), using p21 Waf1/Cip1 (12D1) Rabbit mAb #2947 and α-Tubulin (11H10) Rabbit mAb #2125. The p21 Waf1/Cip1 (12D1) Rabbit mAb confirms silencing of p21 Waf1/Cip1 expression and α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of p21 Waf1/Cip1 siRNA.

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IHC-P (paraffin) Image 28

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using HES1 (D6P2U) Rabbit mAb.

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IHC-P (paraffin) Image 29

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Cyclin D3 (DCS22) Mouse mAb.

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IHC-P (paraffin) Image 30

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Notch1 (D1E11) XP® Rabbit mAb.

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IHC-P (paraffin) Image 31

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using RBPSUH (D10A4) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

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Chromatin IP Image 32

Chromatin immunoprecipitations were performed with cross-linked chromatin from CUTLL1 cells cultured in media with γ-secretase inhibitor (1 μM) for 3 days and then washed and cultured in fresh media for 3 hr and either MAML1 (D3K7B) 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 DTX1 intron 3 primers, SimpleChIP® Human HES4 Promoter Primers #7273, 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.

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Western Blotting Image 33

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® c-Myc siRNA I #6341 (+), using c-Myc (D3N8F) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The c-Myc (D3N8F) Rabbit mAb confirms silencing of c-Myc expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.

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IP Image 34

Immunoprecipitation of p21 from human umbillical vein endothelial cells (HUVECs) using p21 Waf1/Cip1 (12D1) Rabbit mAb. Western blot detection was performed using the same antibody.

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IHC-P (paraffin) Image 35

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Cyclin D3 (DCS22) Mouse mAb.

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IHC-P (paraffin) Image 36

Immunohistochemical analysis of paraffin-embedded A2780 (left), Jurkat (center) and RL (right) cell pellets using Notch1 (D1E11) XP® Rabbit mAb. Both A2780 and Jurkat express Notch1, but only Jurkat cells have cleaved Notch1, while RL cells express very low or no Notch1.

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IHC-P (paraffin) Image 37

Immunohistochemical analysis of paraffin-embedded E18.5 mouse lung, Rbpjk F/+ Shh+/+ (wild type, left) or Rbpjk F/- Shhcre/+ (Rbpjk conditional knock out, right), using RBPSUH (D10A4) XP® Rabbit mAb. Note lack of staining in the bronchial epithelial cells in the conditional knock out tissue (right). Tissue courtesy of Dr. Wellington Cardosa, Boston University School of Medicine.

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Flow Cytometry Image 38

Flow cytometric analysis of HT29 cells using Myc Rabbit mAb, treated with JQ1 (1uM, 72 hours at 37C) (blue) and untreated (green). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) was used as a secondary antibody.

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IHC-P (paraffin) Image 39

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using p21 Waf1/Cip1 (12D1) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

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IHC-P (paraffin) Image 40

Immunohistochemical analysis of paraffin-embedded CUTLL1 cell pellets, control (left) or Compound E-treated (right), using HES1 (D6P2U) Rabbit mAb.

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IHC-P (paraffin) Image 41

Immunohistochemical analysis of paraffin-embedded human stomach adjacent to MALT (mucosa-associated lymphoid tissue) lymphoma using Notch1 (D1E11) XP® Rabbit mAb.

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IHC-P (paraffin) Image 42

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using RBPSUH (D10A4) XP® Rabbit mAb.

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IF-IC Image 43

Confocal immunofluorescent analysis of HT-29 cells, untreated (left) or treated with bromodomain inhibitor JQ1 (1 μM, 72 hr; right), using c-Myc (D3N8F) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red).

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IHC-P (paraffin) Image 44

Immunohistochemical analysis of paraffin-embedded HeLa cells, transfected with SignalSilence® Control siRNA (Unconjugated) #6568 (left) or SignalSilence® p21 Waf1/Cip1 siRNA II #6558 (right), using p21 Waf1/Cip1 (12D1) Rabbit mAb.

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Chromatin IP Image 45

CUTLL1 cells were cultured in media with γ-secretase inhibitor (1 μM) for 3 days and then either harvested immediately (left panel) or washed and cultured in fresh media for 3 hr (right panel). Chromatin immunoprecipitations were performed with cross-linked chromatin from cells and Notch1 (D1E11) 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 human HES1 promoter primers, SimpleChIP® Human HES4 Promoter Primers #7273, 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.

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IHC-P (paraffin) Image 46

Immunohistochemical analysis of paraffin-embedded mouse lymph node using RBPSUH (D10A4) XP® Rabbit mAb.

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Flow Cytometry Image 47

Flow cytometric analysis of HeLa cells (red) and MCF7 cells (blue), using p21 Waf1/Cip1 (12D1) Rabbit mAb.

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IF-IC Image 48

Confocal immunofluorescent analysis of MCF7 cells using p21 Waf1/Cip1 (12D1) Rabbit mAb (red) and Phospho-Histone H3 (Ser10) (6G3) Mouse mAb #9706 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Notch1 (D1E11) XP® Rabbit mAb 3608 20 µl
  • WB
  • IP
  • IHC
  • ChIP
H M R 120, 300 Rabbit IgG
Cleaved Notch1 (Val1744) (D3B8) Rabbit mAb 4147 20 µl
  • WB
  • IP
  • ChIP
H M R 110 Rabbit IgG
RBPSUH (D10A4) XP® Rabbit mAb 5313 20 µl
  • WB
  • IHC
  • ChIP
H M R Mk 61 Rabbit IgG
MAML1 (D3K7B) Rabbit mAb 12166 20 µl
  • WB
  • IP
  • ChIP
H M R 130 Rabbit IgG
c-Myc (D3N8F) Rabbit mAb 13987 20 µl
  • WB
  • IF
  • F
  • ChIP
H M R Mk 57-65 Rabbit IgG
p21 Waf1/Cip1 (12D1) Rabbit mAb 2947 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H Mk 21 Rabbit IgG
HES1 (D6P2U) Rabbit mAb 11988 20 µl
  • WB
  • IP
  • IHC
H M R Mk 30 Rabbit IgG
Cyclin D3 (DCS22) Mouse mAb 2936 20 µl
  • WB
  • IHC
H M R 31 Mouse IgG1
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

The Notch Activated Targets Antibody Sampler Kit provides an economical means of detecting target proteins of activated Notch. The kit contains enough primary antibody to perform four western blot experiments per primary antibody.

Notch1 (D1E11) XP® Rabbit mAb detects intracellular epitopes between 2400 and 2500 amino acids of human Notch1. It recognizes both the full-length (~300 kDa) and the NTM region (~120 kDa). The antibody cannot detect the extracellular (ligand-binding) domain of Notch1 following cleavage at the S2 site by ADAM-type metalloproteases. Cleaved Notch1 (V1744) (D3B8) Rabbit mAb detects endogenous levels of the Notch1 intracellular domain (NICD) only when released by cleavage between Gly1753 and Val1754 (equivalent to Gly1743/Val1744 of murine notch1). The antibody does not recognize full-length Notch1 or Notch1 cleaved at other positions. The size of the NICD varies among cell lines due to mutations in the Notch1 C-terminus. RBPSUH (D10A4) XP® Rabbit mAb recognizes endogenous levels of total RBPSUH protein. MAML1 (D3K7B) Rabbit mAb recognizes endogenous levels of total MAML1 protein. This antibody does not detect MAML2 or MAML3. c-Myc (D3N8F) Rabbit mAb recognizes endogenous levels of total c-Myc protein. p21 Waf1/Cip1 (12D1) Rabbit mAb detects endogenous levels of total p21 protein. The antibody does not cross-react with other CDK inhibitors. HES1 (D6P2U) Rabbit mAb recognizes endogenous levels of total HES1 protein. Cyclin D3 (DCS22) Mouse mAb detects endogenous levels of total cyclin D3 protein. The antibody does not cross-react with cyclin D1 or cyclin D2. 

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro2438 of human Notch1, the Val1754 cleavage site in human Notch1 (equivalent to Val1744 in mouse Notch1), residues surrounding Gln110 of human RBPSUH protein, residues surrounding Asp269 of human MAML1 protein, a central region within human c-Myc protein, residues near the carboxy-terminus of human p21,  recombinant protein specific to human HES1 protein, residues 241-260 of  recombinant human cyclin D3. Antibodies are purified by protein A and peptide affinity chromatography.

Notch proteins (Notch1-4) are a family of transmembrane receptors that play important roles in development and the determination of cell fate (1). Mature Notch receptors are processed and assembled as heterodimeric proteins, with each dimer comprised of a large extracellular ligand-binding domain, a single-pass transmembrane domain, and a smaller cytoplasmic subunit (Notch intracellular domain, NICD) (2). Binding of Notch receptors to ligands of the Delta-Serrate-Lag2 (DSL) family triggers heterodimer dissociation, exposing the receptors to proteolytic cleavages; these result in release of the NICD, which translocates to the nucleus and activates transcription of downstream target genes (3,4).  RBPSUH (Recombining Binding Protein, SUppressor of Hairless), is the DNA-binding component of the transcription complex regulated by canonical Notch signaling. Binding of Notch with RBPSUH activates a transcription activation complex that includes Mastermind-like (MAML) proteins, leading to transcriptional activation of Notch target genes (5-7). The NICD binds and activates c-Myc which functions as a transcriptional regulator with roles in various aspects of cell behavior including proliferation, differentiation and apoptosis (8).  The tumor suppressor protein p21 Waf1/Cip1 acts as an inhibitor of cell cycle progression. The NICD-RBPSUH complex binds and activates p21 for transcription (15).  HES1 (Hairy and Enhancer of Split 1) is one of seven members of the HES family of basic helix-loop-helix (bHLH) transcription factors that is particularly well known as a repressive mediator of the canonical Notch signaling pathway (10). HES1 plays a key role in mediating Notch-dependent T cell lineage commitment (11), and has been reported to be an essential mediator of Notch-induced T cell acute lymphoblastic leukemia (T-ALL) (11,12). The active complex of cyclin D/CDK4 targets the retinoblastoma protein for phosphorylation, allowing the release of E2F transcription factors that activate G1/S-phase gene expression (13).  Transcription of cyclin D is in part regulated by the NICD binding to the promoter region of cyclin D (14).

  1. Li, X. and von Boehmer, H. (2011) ISRN Hematol 2011, 921706.
  2. Niimi, H. et al. (2007) J Cell Biol 176, 695-707.
  3. Artavanis-Tsakonas, S. et al. (1999) Science 284, 770-6.
  4. Wu, L. et al. (2002) Mol. Cell Biol. 22, 7688-7700.
  5. Baudino, T.A. and Cleveland, J.L. (2001) Mol Cell Biol 21, 691-702.
  6. Chan, Y.M. and Jan, Y.N. (1998) Cell 94, 423-6.
  7. Lin, S.E. et al. (2002) J. Biol. Chem. 277, 50612-50620.
  8. Schroeter, E.H. et al. (1998) Nature 393, 382-6.
  9. Kitagawa, M. et al. (2001) Mol. Cell Biol. 21, 4337-4346.
  10. Flores-Rozas, H. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 8655-8659.
  11. Kobayashi, T. and Kageyama, R. (2010) Genes Cells 15, 689-98.
  12. Lukas, J. et al. (1996) Mol Cell Biol 16, 6917-25.
  13. Rand, M.D. et al. (2000) Mol Cell Biol 20, 1825-35.
  14. Wendorff, A.A. et al. (2010) Immunity 33, 671-84.
  15. Espinosa, L. et al. (2010) Cancer Cell 18, 268-81.
Entrez-Gene Id
896 , 3280 , 9794 , 4609 , 4851 , 1026 , 3516
Swiss-Prot Acc.
P30281 , Q14469 , Q92585 , P01106 , P46531 , P38936 , Q06330
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.

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