Revision 3

#73271Store 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

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

78

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q13546

Entrez-Gene Id:

8737

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:200
Immunohistochemistry (Paraffin) 1:300 - 1:1200
Immunofluorescence (Immunocytochemistry) 1:800

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.

For a carrier free (BSA and azide free) version of this product see product #81295.

Specificity / Sensitivity

RIP (E8S7U) XP® Rabbit mAb recognizes endogenous levels of full-length RIP protein as well as the C-terminal fragment produced by caspase cleavage.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg424 of human RIP 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).
Necroptosis, a regulated pathway for necrotic cell death, is triggered by a number of inflammatory signals including cytokines in the tumor necrosis factor (TNF) family, pathogen sensors such as toll-like receptors (TLRs), and ischemic injury (9,10). The process is negatively regulated by caspases and is initiated through a complex containing the RIP and RIP3 kinases, typically referred to as the necrosome. Necroptosis is inhibited by a small molecule inhibitor of RIP, necrostatin-1 (Nec-1) (11). Research studies show that necroptosis contributes to a number of pathological conditions, and Nec-1 has been shown to provide neuroprotection in models such as ischemic brain injury (12). RIP is phosphorylated at several sites within the kinase domain that are sensitive to Nec-1, including Ser14, Ser15, Ser161, and Ser166 (13).

  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. Christofferson, D.E. and Yuan, J. (2010) Curr Opin Cell Biol 22, 263-8.
  10. Kaczmarek, A. et al. (2013) Immunity 38, 209-23.
  11. Degterev, A. et al. (2008) Nat Chem Biol 4, 313-21.
  12. Degterev, A. et al. (2005) Nat Chem Biol 1, 112-9.
  13. Ofengeim, D. and Yuan, J. (2013) Nat Rev Mol Cell Biol 14, 727-36.

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)

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.
SignalStain is a registered trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
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
#73271

RIP (E8S7U) XP® Rabbit mAb

Western Blotting Image 1: RIP (E8S7U) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from HT-29 and HT-29 RIPK1 knockout (-/-) cells using RIP (E8S7U) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). HT-29 RIPK1 knockout cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston, MA.
Western Blotting Image 2: RIP (E8S7U) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using RIP (E8S7U) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 3: RIP (E8S7U) XP® Rabbit mAb Expand Image
Western blot analysis of Jurkat cells, untreated (-) or treated with Etoposide #2200 (25 μM, 20 hr; +), using RIP (E8S7U) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb (lower).
Immunoprecipitation Image 1: RIP (E8S7U) XP® Rabbit mAb Expand Image
Immunoprecipitation of RIP from MCF7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is RIP (E8S7U) XP® Rabbit mAb. Western blot analysis was performed using RIP (E8S7U) XP® Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
Immunohistochemistry Image 1: RIP (E8S7U) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using RIP (E8S7U) XP® Rabbit mAb.
Immunohistochemistry Image 2: RIP (E8S7U) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using RIP (E8S7U) XP® Rabbit mAb.
Immunohistochemistry Image 3: RIP (E8S7U) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human non-Hodgkin lymphoma using RIP (E8S7U) XP® Rabbit mAb.
Immunohistochemistry Image 4: RIP (E8S7U) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human ductal gastric adenocarcinoma using RIP (E8S7U) XP® Rabbit mAb.
Immunohistochemistry Image 5: RIP (E8S7U) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human colon carcinoma (left), squamous cell lung carcinoma (middle) or esophageal carcinoma (right) using RIP (E8S7U) XP® Rabbit mAb (top) or RIP Rabbit mAb (bottom). These two antibodies detect independent, unique epitopes on human RIP. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.
Immunohistochemistry Image 6: RIP (E8S7U) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using RIP (E8S7U) XP® Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Immunohistochemistry Image 7: RIP (E8S7U) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human ovarian clear cell carcinoma using RIP (E8S7U) XP® Rabbit mAb.
Immunohistochemistry Image 8: RIP (E8S7U) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded HT-29 cell pellet (left) or HT-29 RIPK1 knockout (-/-) cell pellet (right) using RIP (E8S7U) XP® Rabbit mAb.
Immunofluorescence Image 1: RIP (E8S7U) XP® Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HT-29 cells, either wild-type (left, positive) or RIPK1 knockout (-/-) (right, negative), using RIP (E8S7U) 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).