Revision 3
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, IF-IC

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
Immunofluorescence (Immunocytochemistry) 1:800 - 1:3200

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

Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb recognizes endogenous levels of RIP3 protein only when phosphorylated at Ser227. A band is also detected at 30 kDa that appears to be a cleavage product of RIP3.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Ser227 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). 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. 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.
  15. Chen, W. et al. (2013) J Biol Chem 288, 16247-61.

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 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.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

限制使用

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专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品, (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Revision 3
#93654

Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb

Western Blotting Image 1: Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb Expand Image
对未经处理 (-) 或已经以下联合处理方法处理的 HT-29 细胞进行蛋白质印迹分析:Z-VAD(20 μM,先于其他化合物 30 分钟添加;+)、Human Tumor Necrosis Factor-α #8902(hTNF-α,20 ng/ml,7 小时;+)和 SM-164(100 nM,7 小时;+),使用 Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb。膜不用(左图)或用牛小肠磷酸酶处理(CIP;右图),以确定磷酸特异性。
Western Blotting Image 2: Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb Expand Image
对未经处理 (-) 或已经以下联合处理方法处理的 HT-29 细胞进行蛋白质印迹分析:Z-VAD(20 μM,先于其他混合物 30 分钟添加;+)、人 Tumor Necrosis Factor-α #8902(hTNF-α, 20 ng/ml,7 小时;+)、SM-164(100 nM,7 小时;+)和 necrostatin-1(Nec-1,50 μM, 7小时;+),使用 Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb(上图),RIP3 (E1Z1D) Rabbit mAb #13526(中图)或 β-Actin (D6A8) Rabbit mAb #8457(下图)。
Western Blotting Image 3: Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb Expand Image
对未经处理 (-) 或已经以下联合处理方法处理的 HT-29 细胞或 HT-29 RIPK1 KO 细胞进行蛋白质印迹分析:Z-VAD(20 μM,先于其他混合物 30 分钟添加;+)、人 Tumor Necrosis Factor-α #8902(hTNF-α,20 ng/ml, 7 小时;+)和 SM-164(100 nM,7 小时;+)使用 Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb(上图),RIP3 (E1Z1D) Rabbit mAb #13526(中图)或 β-Actin (D6A8) Rabbit mAb #8457(下图)。HT-29 RIPK1 KO 细胞由马萨诸塞州波士顿哈佛大学医学院的 Junying Yuan 博士惠赠。
Immunofluorescence Image 1: Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb Expand Image
使用 Phospho-RIPK3 (Ser227) (D6W2T) Rabbit mAb(绿色)对未处理的(左图)、经 Z-VAD(20 μM,30 分钟)预处理后再经 SM-164 (100 nM) 和 Human Tumor Necrosis Factor-α (hTNF-α) #8902(20 ng/mL,6 小时;中间)处理,或经 Z-VAD 预处理后再经 SM-164 和 hTNF-α 处理,随后经 λ 磷酸酶后处理(右图)的 HT-29 细胞进行共聚焦免疫荧光分析。肌动蛋白微丝由 DyLight 554 Phalloidin #13054(红色)标记。蓝色伪彩 = DRAQ5® #4084(DNA 荧光染料)。