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54105
PhosphoPlus® RIP3 (Ser227) Antibody Duet
Primary Antibodies

PhosphoPlus® RIP3 (Ser227) Antibody Duet #54105

Western Blotting Image 1

Western blot analysis of extracts from various cell lines using RIP3 (E1Z1D) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 2

Western blot analysis of HT-29 cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), and SM-164 (100 nM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb. To confirm phospho-specificity, membranes were either untreated (left) or treated with Calf Intestinal Phosphatase (CIP; right).

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Western Blotting Image 3

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length human RIP3 protein (hRIP3; +), using RIP3 (E1Z1D) Rabbit mAb.

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Western Blotting Image 4

Western blot analysis of HT-29 cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb (upper), RIP3 (E1Z1D) Rabbit mAb #13526 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 5

Western blot analysis of HT-29 cells or HT-29 RIPK1 KO cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), and SM-164 (100 nM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb (upper), RIP3 (E1Z1D) Rabbit mAb #13526 (middle) or β-Actin (D6A8) Rabbit mAb #8457 (lower). HT-29 RIPK1 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston, MA.

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IF-IC Image 6

Confocal immunofluorescent analysis of HT-29 cells, untreated (left), pre-treated with Z-VAD (20 μM, 30 min) followed by treatment with SM-164 (100 nM) and Human Tumor Necrosis Factor-α (hTNF-α) #8902 (20 ng/mL, 6 hr; center), or pre-treated with Z-VAD followed by treatment with SM-164 and hTNF-α and post-processed with λ-phosphatase (right), using Phospho-RIPK3 (Ser227) (D6W2T) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
RIP3 (E1Z1D) Rabbit mAb 13526 100 µl
  • WB
  • IP
H 46-62 Rabbit IgG
Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb 93654 100 µl
  • WB
  • IF
H 46-62 Rabbit IgG

PhosphoPlus® Duets from Cell Signaling Technology (CST) provide a means to assess protein activation status. Each Duet contains an activation-state and total protein antibody to your target of interest. These antibodies have been selected from CST's product offering based upon superior performance in specified applications.

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). Subsequently, it has 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). In mice, RIP3 is phosphorylated at Thr231 and Ser232, leading to association with MLKL and necroptosis (15).

  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. Chen, W. et al. (2013) J Biol Chem 288, 16247-61.
  10. Yu, P.W. et al. (1999) Curr Biol 9, 539-42.
  11. Sun, X. et al. (1999) J Biol Chem 274, 16871-5.
  12. Zhang, D.W. et al. (2009) Science 325, 332-6.
  13. He, S. et al. (2009) Cell 137, 1100-11.
  14. Cho, Y.S. et al. (2009) Cell 137, 1112-23.
  15. Sun, L. et al. (2012) Cell 148, 213-27.
Entrez-Gene Id
11035
Swiss-Prot Acc.
Q9Y572
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PhosphoPlus is a trademark of Cell Signaling Technology, Inc.

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