Western blot analysis of extracts from various cell types using ADAM9 (D64B5) Rabbit mAb.
Western blot analysis of extracts from COS cells, untransfected or transiently transfected with a construct expressing rat DLL1 protein, using DLL1 Antibody.
Western blot analysis of COS cell extracts, untransfected or transiently transfected with a construct expressing rat DLL3 protein, using DLL3 (G93) Antibody.
Western blot analysis of extracts from HUVEC and COS cells, untransfected or transiently transfected with a construct expressing human DLL4, using DLL4 Antibody.
Western blot analysis of total cell lysates from HepG2 and LNCaP cells, using Jagged1 (28H8) Rabbit mAb.
Western blot analysis of total cell lysates from HeLa, SK-OV-3 and SR cells using Jagged2 (C23D2) Rabbit mAb.
Western blot analysis of extracts from various cell lines using Numb (C29G11) Rabbit mAb.
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.
Western blot analysis of extracts from various cell lines using RBPSUH (D10A4) XP® Rabbit mAb.
Western blot analysis of extracts from Raji and Jurkat cells, untreated or treated with peptide N-glycosidase F (PNGase F), using TACE (D22H4) Rabbit 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.
Immunoprecipitation/western blot analysis of lysates from HeLa cells. Lane 1 contains lysate input (10%), lane 2 was immunoprecipitated with non-specific rabbit IgG (#3900), lane 3 was immunoprecipitated with Numb (C29G11) Rabbit mAb #2756. Western blot analysis was performed using Numb (C29G11) Rabbit mAb #2756.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using RBPSUH (D10A4) XP® Rabbit mAb.
Flow cytometric analysis of A-204 cells using Numb (C29G11) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).
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).
Confocal immunofluorescent analysis of HeLa cells using Numb (C29G11) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
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.
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using RBPSUH (D10A4) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded mouse lymph node using RBPSUH (D10A4) XP® Rabbit mAb.
|ADAM9 (D64B5) Rabbit mAb 4151||20 µl||
||H M R Mk||100-115, 75-80||Rabbit IgG|
|DLL1 Antibody 2588||20 µl||
|DLL3 (G93) Antibody 2483||20 µl||
|DLL4 Antibody 2589||20 µl||
|Jagged1 (28H8) Rabbit mAb 2620||20 µl||
||H M||180||Rabbit IgG|
|Jagged2 (C23D2) Rabbit mAb 2210||20 µl||
||H R||150||Rabbit IgG|
|Numb (C29G11) Rabbit mAb 2756||20 µl||
||H M R Mk||72, 74||Rabbit IgG|
|RBPSUH (D10A4) XP® Rabbit mAb 5313||20 µl||
||H M R Mk||61||Rabbit IgG|
|TACE (D22H4) Rabbit mAb 6978||20 µl||
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The Notch Receptor Interaction Antibody Sampler Kit provides an economical means to evaluate Notch signaling. The kit contains enough primary antibody to perform two western blots per primary.
ADAM9 (D64B5) Rabbit mAb, DLL4 Antibody, Jagged1 (28H8) Rabbit mAb, Jagged2 (C23D2) Rabbit mAb, Numb (C29G11) Rabbit mAb, RBPSUH (D10A4) XP® Rabbit mAb, and TACE (D22H4) Rabbit mAb recognize endogenous levels of total respecitive protein. DLL1 Antibody recognizes only transfected levels of DLL1 protein. It does not recognize transfected levels of rat DLL3 or human DLL4. DLL3 (G93) Antibody recognizes only transfected levels of DLL3 protein. It does not recognize transfected levels of rat DLL1 or human DLL4. Jagged1 (28H8) Rabbit mAb does not cross-react with Jagged2. Jagged2 (C23D2) Rabbit mAb does not cross-react with Jagged1.
Monoclonal antibodies are produced by immunizing animals either with a recombinant protein specific to the amino terminus of human TACE protein or with a synthetic peptide corresponding to residues surrounding Glu1140 (intracellular region) of human Jagged1 protein, residues surrounding Ala117 of human Jagged2 protein, residues surrounding Ala570 of human Numb protein, or residues near the carboxy terminus of human ADAM9 protein or residues surrounding Gln110 of human RBPSUH protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala627 of human DLL1 protein, residues surrounding Gly93 of mouse DLL3 protein, residues surrounding Leu617 of human DLL4 protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.
Notch signaling is activated upon engagement of the Notch receptor with its ligands, the Delta, Serrate, Lag2 (DSL) single-pass type I membrane proteins. DSL proteins contain multiple EGF-like repeats and a DSL domain that is required for binding to Notch (1,2). Five DSL proteins have been identified in mammals: Jagged1, Jagged2, Delta-like (DLL) 1, 3, and 4 (3). Ligand binding to the Notch receptor results in two sequential proteolytic cleavages of the receptor by the ADAM protease and the γ-secretase complex. The intracellular domain of Notch is released and then translocates to the nucleus where it activates transcription. Notch ligands may also be processed in a similiar manner, suggesting bi-directional signaling through receptor-ligand interactions (4-6).
TNF-α converting enzyme (TACE), also known as ADAM17, is a transmembrane metalloprotease that plays a key role in the cleavage of a number cell surface molecules in a process known as “shedding". TACE is abundantly expressed in many adult tissues, but in fetal development, expression is differentially regulated (7). TACE activates Notch in a ligand-independent manner and has been shown to play a role in the development of the Drosophila nervous system (8).
Recombining Binding Protein, SUppressor of Hairless (RBPSUH), also termed RBP-J or CSL, is the DNA-binding component of the transcription complex regulated by canonical Notch signaling. In the absence of Notch activation, RBPSUH suppresses target gene expression through interactions with a co-repressor complex containing histone deacetylase. Upon activation of Notch receptors, the Notch intracellular domain (NICD) translocates to the nucleus and binds to RBPSUH. This displaces the co-repressor complex and replaces it with a transcription activation complex that includes Mastermind-like (MAML) proteins and histone acetylase p300, leading to transcriptional activation of Notch target genes (9-11).
Numb contains an amino-terminal phosphotyrosine-binding (PTB) domain and carboxy-terminal endocytic binding motifs for α-adaptin and EH (Eps15 homology) domain-containing proteins, indicating a role in endocytosis (12,13). There are four mammalian Numb splicing isoforms that are differentially expressed and may have distinct functions (14-16). Numb acts as a negative regulator of Notch signaling by promoting ubiquitination and degradation of Notch (17). The protein is asymmetrically segregated into one daughter cell during cell division, producing two daughter cells with different responses to Notch signaling and different cell fates (18,19).
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