Revision 3

#10188Store at -20C

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.
Applications:

WB, IP, IHC-P, IF-IC, FC-FP

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

46-62

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q9Y572

Entrez-Gene Id:

11035

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:50
Immunohistochemistry (Paraffin) 1:50 - 1:200
Immunofluorescence (Immunocytochemistry) 1:100 - 1:400
Flow Cytometry (Fixed/Permeabilized) 1:400 - 1:1600

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.

Specificity / Sensitivity

RIP3 (E7A7F) XP® Rabbit mAb recognizes endogenous levels of total RIP3 protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human RIP3 protein.

Background

The receptor-interacting protein (RIP) family of serine-threonine kinases (RIP, RIP2, RIP3, and RIP4) are important regulators of cellular stress that trigger pro-survival and inflammatory responses through the activation of NF-κB, as well as pro-apoptotic pathways (1). In addition to the kinase domain, RIP contains a death domain responsible for interaction with the death domain receptor Fas and recruitment to TNF-R1 through interaction with TRADD (2,3). RIP-deficient cells show a failure in TNF-mediated NF-κB activation, making the cells more sensitive to apoptosis (4,5). RIP also interacts with TNF-receptor-associated factors (TRAFs) and can recruit IKKs to the TNF-R1 signaling complex via interaction with NEMO, leading to IκB phosphorylation and degradation (6,7). Overexpression of RIP induces both NF-κB activation and apoptosis (2,3). Caspase-8-dependent cleavage of the RIP death domain can trigger the apoptotic activity of RIP (8).
Receptor-interacting protein 3 (RIP3) was originally found to interact with RIP and the TNF receptor complex to induce apoptosis and activation of NF-κB (9,10). It has subsequently been shown that the association between RIP and RIP3 is a key component of a signaling pathway that results in programmed necrosis (necroptosis), a necrotic-like cell death induced by TNF in the presence of caspase inhibitors (11-13). RIP3 is phosphorylated at Ser227 and targets the phosphorylation of mixed lineage kinase domain-like protein (MLKL), which is critical for necroptosis (14).

  1. Meylan, E. and Tschopp, J. (2005) Trends Biochem Sci 30, 151-9.
  2. Hsu, H. et al. (1996) Immunity 4, 387-96.
  3. Stanger, B.Z. et al. (1995) Cell 81, 513-23.
  4. Ting, A.T. et al. (1996) EMBO J 15, 6189-96.
  5. Kelliher, M.A. et al. (1998) Immunity 8, 297-303.
  6. Devin, A. et al. (2000) Immunity 12, 419-29.
  7. Zhang, S.Q. et al. (2000) Immunity 12, 301-11.
  8. Lin, Y. et al. (1999) Genes Dev 13, 2514-26.
  9. Yu, P.W. et al. (1999) Curr Biol 9, 539-42.
  10. Sun, X. et al. (1999) J Biol Chem 274, 16871-5.
  11. Zhang, D.W. et al. (2009) Science 325, 332-6.
  12. He, S. et al. (2009) Cell 137, 1100-11.
  13. Cho, Y.S. et al. (2009) Cell 137, 1112-23.
  14. Sun, L. et al. (2012) Cell 148, 213-27.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 3
#10188

RIP3 (E7A7F) XP® Rabbit mAb

Western Blotting Image 1: RIP3 (E7A7F) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using RIP3 (E7A7F) XP® Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Absence of signal in HeLa cells is predicted from published reports of epigenetic loss of expression and confirms the specificity of the antibody for RIP3.
Immunoprecipitation Image 1: RIP3 (E7A7F) XP® Rabbit mAb Expand Image
Immunoprecipitation of RIP3 protein from THP-1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is RIP3 (E7A7F) XP® Rabbit mAb. Western blot analysis was performed using RIP3 (E7A7F) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
Immunohistochemistry Image 1: RIP3 (E7A7F) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human colon carcinoma (left), hepatocellular carcinoma (middle), or normal thymus (right) using RIP3 (E7A7F) XP® Rabbit mAb (top) or RIP3 Rabbit mAb (bottom). These two antibodies detect independent, unique epitopes on human RIP3 protein. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.
Immunohistochemistry Image 2: RIP3 (E7A7F) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using RIP3 (E7A7F) XP® Rabbit mAb.
Immunohistochemistry Image 3: RIP3 (E7A7F) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded HT-29 cell pellet (left, high-expressing) or HeLa cell pellet (right, low-expressing) using RIP3 (E7A7F) XP® Rabbit mAb.
Immunohistochemistry Image 4: RIP3 (E7A7F) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded normal human colon using RIP3 (E7A7F) XP® Rabbit mAb.
Immunohistochemistry Image 5: RIP3 (E7A7F) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using RIP3 (E7A7F) XP® Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Immunohistochemistry Image 6: RIP3 (E7A7F) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human esophageal adenocarcinoma using RIP3 (E7A7F) XP® Rabbit mAb.
Immunohistochemistry Image 7: RIP3 (E7A7F) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human squamous cell carcinoma of the tonsil using RIP3 (E7A7F) XP® Rabbit mAb.
Immunohistochemistry Image 8: RIP3 (E7A7F) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using RIP3 (E7A7F) XP® Rabbit mAb.
Immunofluorescence Image 1: RIP3 (E7A7F) XP® Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HT-29 cells (left, positive) or HeLa cells (right, negative) using RIP3 (E7A7F) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
Flow Cytometry Image 1: RIP3 (E7A7F) XP® Rabbit mAb Expand Image
Flow cytometric analysis of SU-DHL-4 cells (blue, negative) and THP-1 cells (green, positive) using RIP3 (E7A7F) XP® Rabbit mAb (solid lines) or a 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.