ProSci

RIP3 Antibody

Product Code:
 
PSI-2283
Product Group:
 
Primary Antibodies
Supplier:
 
ProSci
Host Type:
 
Rabbit
Antibody Isotype:
 
IgG
Antibody Clonality:
 
Polyclonal
Regulatory Status:
 
RUO
Target Species:
  • Mouse
  • Rat
Applications:
  • Enzyme-Linked Immunosorbent Assay (ELISA)
  • Immunohistochemistry- Paraffin Embedded (IHC-P)
  • Immunoprecipitation (IP)
  • Western Blot (WB)
1 / 15
<strong>Figure 1 Western Blot Validation in Mouse Cell lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: RIP3 2283, (0.5 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
2 / 15
<strong>Figure 2 Western Blot Validation in C2C12 Cells Mouse</strong><br> Loading: 15 μg of lysates per lane. Antibodies: RIP3 2283, 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. Lane 1: 2283, 0.1 μg/mL in the presence of peptide blocking Lane 2: 2283, 0.1 μg/mL Lane 3: 2283, 0.2 μg/mL Lane 4: 2283, 0.5 μg/mL
3 / 15
<strong>Figure 3 Western Blot Validation in Rat Thymus</strong><br> Loading: 15 ?g of lysates per lane. Antibodies: RIP3 2283, 0.5 μg/mL, 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
4 / 15
<strong>Figure 4 Immunohistochemistry Validation of RIP3 in Mouse Lung </strong><br>
5 / 15
<strong>Figure 5 Immunohistochemistry Validation of RIP3 in Mouse Thymus </strong><br> Immunohistochemical analysis of paraffin-embedded mouse thymus tissue using anti-RIP3 antibody (2283) at 1 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
6 / 15
<strong>Figure 6 Immunohistochemistry Validation of RIP3 in Rat Thymus </strong><br> Immunohistochemical analysis of paraffin-embedded rat thymus tissue using anti-RIP3 antibody (2283) at 1 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
7 / 15
<strong>Figure 7 KO Validation of RIP3 in RIP3 KO Mice (He et al., 2009) </strong><br> Western blot analysis of RIP3 with anti-RIP3 antibodies shows disrupted RIP3 expression in pancreas, liver spleen thymus and lung of RIP3 KO mice. Cerulein treatment upregulated RIP3 expression in the pancreas of WT mice.
8 / 15
<strong>Figure 8 Immunohistochemistry Validation of RIP3 in Fadd KO mice (Zhang et al., 2011) </strong><br> RIP3 expression detected by anti-RIP3 antibodies (2283) was decreased in Fadd KO mice as compared to WT mice.
9 / 15
<strong>Figure 9 KO Validation of RIP3 in MEF Cells (He et al., 2012) </strong><br> Western blot analysis of RIP3 with anti-RIP3 antibodies shows disrupted RIP3 expression in RIP3 KO MEFs, but not in WT MEF cells.
10 / 15
<strong>Figure 10 KD Validation of RIP3 in L929 Cells (Narayan et al., 2012) </strong><br> Immunoblot analysis of RIP3 with anti-RIP3 antibodies (2283) shows RIP3 expression was disrupted in RIP3 knockdown L929 cells.
11 / 15
<strong>Figure 11 Regulated Expression Validation of RIP3 in Macrophages (Li et al., 2016) </strong><br> Confocal microscopy shows colocalization of RIPK1 (green) and RIPK3 (red) in PM? and RIPK3 was detected by anti-RIPK3 antibodies (2283). PF or BCM suppressed LPS-induced upregulation of RIP3.
12 / 15
<strong>Figure 12 Overexpression Validation of RIP3 in Cancer Cell Lines (Yang et al., 2017) </strong><br> The cancer cell lines were stably expressing flag-tagged RIP3 and RIP expression was detected by anti-RIP3 antibodies (2283) in RIP3-overexpressed cells.
13 / 15
<strong>Figure 13 Induced Expression of RIP3 by Acetaminophen in WT and RIP3 KO Mice (Ramachandran et al., 2013) </strong><br> Wild-type and RIP3 KO mice were treated with300 mg/kg acetaminophen or saline. RIP3 expression detected by anti-RIP3 antibodies (2283) were up-regulated after acetaminophen treatment in the liver, while this effect was not observed in RIP3 KO mice.
14 / 15
<strong>Figure 14 IP Validation of RIP3 in HT29 Cells (Li et al., 2012) </strong><br> RIP3 complexes isolated by immunoprecipitation with anti-RIP3 antibodies (2283) from HT-29 cells treated with T+Z+L after lysis in regular lysis buffer or buffer containing the indicated amount of urea or NaOH.
15 / 15
<strong>Figure 15 Immunogold EM Validation in MEF Cells (Li et al., 2012) </strong><br> Clustering of RIP3 in necrotic MEFs shown by immunogold EM with anti-RIP3 antibodies (2283). Scale bars, 100 nm (RIP3).
❮❯

<strong>Figure 1 Western Blot Validation in Mouse Cell lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: RIP3 2283, (0.5 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 2 Western Blot Validation in C2C12 Cells Mouse</strong><br> Loading: 15 μg of lysates per lane. Antibodies: RIP3 2283, 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. Lane 1: 2283, 0.1 μg/mL in the presence of peptide blocking Lane 2: 2283, 0.1 μg/mL Lane 3: 2283, 0.2 μg/mL Lane 4: 2283, 0.5 μg/mL
<strong>Figure 3 Western Blot Validation in Rat Thymus</strong><br> Loading: 15 ?g of lysates per lane. Antibodies: RIP3 2283, 0.5 μg/mL, 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 4 Immunohistochemistry Validation of RIP3 in Mouse Lung </strong><br>
<strong>Figure 5 Immunohistochemistry Validation of RIP3 in Mouse Thymus </strong><br> Immunohistochemical analysis of paraffin-embedded mouse thymus tissue using anti-RIP3 antibody (2283) at 1 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 6 Immunohistochemistry Validation of RIP3 in Rat Thymus </strong><br> Immunohistochemical analysis of paraffin-embedded rat thymus tissue using anti-RIP3 antibody (2283) at 1 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 7 KO Validation of RIP3 in RIP3 KO Mice (He et al., 2009) </strong><br> Western blot analysis of RIP3 with anti-RIP3 antibodies shows disrupted RIP3 expression in pancreas, liver spleen thymus and lung of RIP3 KO mice. Cerulein treatment upregulated RIP3 expression in the pancreas of WT mice.
<strong>Figure 8 Immunohistochemistry Validation of RIP3 in Fadd KO mice (Zhang et al., 2011) </strong><br> RIP3 expression detected by anti-RIP3 antibodies (2283) was decreased in Fadd KO mice as compared to WT mice.
<strong>Figure 9 KO Validation of RIP3 in MEF Cells (He et al., 2012) </strong><br> Western blot analysis of RIP3 with anti-RIP3 antibodies shows disrupted RIP3 expression in RIP3 KO MEFs, but not in WT MEF cells.
<strong>Figure 10 KD Validation of RIP3 in L929 Cells (Narayan et al., 2012) </strong><br> Immunoblot analysis of RIP3 with anti-RIP3 antibodies (2283) shows RIP3 expression was disrupted in RIP3 knockdown L929 cells.
<strong>Figure 11 Regulated Expression Validation of RIP3 in Macrophages (Li et al., 2016) </strong><br> Confocal microscopy shows colocalization of RIPK1 (green) and RIPK3 (red) in PM? and RIPK3 was detected by anti-RIPK3 antibodies (2283). PF or BCM suppressed LPS-induced upregulation of RIP3.
<strong>Figure 12 Overexpression Validation of RIP3 in Cancer Cell Lines (Yang et al., 2017) </strong><br> The cancer cell lines were stably expressing flag-tagged RIP3 and RIP expression was detected by anti-RIP3 antibodies (2283) in RIP3-overexpressed cells.
<strong>Figure 13 Induced Expression of RIP3 by Acetaminophen in WT and RIP3 KO Mice (Ramachandran et al., 2013) </strong><br> Wild-type and RIP3 KO mice were treated with300 mg/kg acetaminophen or saline. RIP3 expression detected by anti-RIP3 antibodies (2283) were up-regulated after acetaminophen treatment in the liver, while this effect was not observed in RIP3 KO mice.
<strong>Figure 14 IP Validation of RIP3 in HT29 Cells (Li et al., 2012) </strong><br> RIP3 complexes isolated by immunoprecipitation with anti-RIP3 antibodies (2283) from HT-29 cells treated with T+Z+L after lysis in regular lysis buffer or buffer containing the indicated amount of urea or NaOH.
<strong>Figure 15 Immunogold EM Validation in MEF Cells (Li et al., 2012) </strong><br> Clustering of RIP3 in necrotic MEFs shown by immunogold EM with anti-RIP3 antibodies (2283). Scale bars, 100 nm (RIP3).

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Further Information

Additional Names:
RIP3 Antibody: Rip3, AW107945, 2610528K09Rik, Rip3, RIP-like protein kinase 3, RIP-3
Application Note:
WB: 0.1-0.5 μg/mL; IHC-P: 5 μg/mL; IF: 20 μg/mL, IP: 20 μg/mL.

Antibody validated: Western Blot in mouse and human samples; Immunohistochemistry in mouse and rat samples; Immunofluorescence in mouse and rat samples; Immunoprecipitation in human samples; Immunogold EM in mouse samples. All other applications and species not yet tested.
Background:
RIP3 Antibody: Certain serine/threonine protein kinases, such as ASK1, RIP, DAP, and ZIP kinases, are mediators of apoptosis. Receptor interacting proteins including RIP and RIP2/RICK mediate apoptosis induced by TNFR1 and Fas, two prototype members in the death receptor family. A novel member in the RIP kinase family was recently identified and designated RIP3. RIP3 contains N-terminal kinase domain but, unlike RIP or RIP2, lacks the C-terminal death or CARD domain. RIP3 binds to RIP and TNFR1, mediates TNFR1 induced apoptosis, and attenuates RIP and TNFR1 induced NF-κB activation. Overexpression of RIP3 induces apoptosis and NF-κB activation. The messenger RNA of RIP3 is expressed in a subset of adult tissues.
Background References:
  • Yu et al. Curr Biol. 1999;9(10):539-42.
  • Sun et al. J Biol Chem. 1999;274(24):16871-5.
  • Pazdernik et al. Mol Cell Bio. 1999; 19(10):6500-8 (WD0102)
Buffer:
RIP3 Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/ml
Conjugate:
Unconjugated
DISCLAIMER:
Optimal dilutions/concentrations should be determined by the end user. The information provided is a guideline for product use. This product is for research use only.
Immunogen:
Anti-RIP3 antibody (2283) was raised against a peptide corresponding to 14 amino acids near the carboxy terminus of murine RIP3.

The immunogen is located within the last 50 amino acids of RIP3.
Isoforms:
Mouse RIP3 has one isoform (486aa, 53kD). Human RIP3 has 3 isoforms, including isoform 1 (518aa, 57kD), isoform 2 (252aa, 28kD) and isoform 3 (231aa, 25kD). Rat RIP3 also has one isoform (478aa, 52kD). 2283 can detect can detect isoforms of mouse and rat.
NCBI Gene ID #:
56532
NCBI Official Name:
receptor-interacting serine-threonine kinase 3
NCBI Official Symbol:
Ripk3
NCBI Organism:
Mus musculus
Physical State:
Liquid
Predicted Molecular Weight:
Predicted: 53kD for mouse RIP3 and 57kD for human RIP3
Observed: 53kD for mouse RIP3 and 57kD for human RIP3
Protein Accession #:
AAF03133
Protein GI Number:
6063101
Purification:
RIP3 Antibody is affinity chromatography purified via peptide column.
Research Area:
Apoptosis
Specificity:
Specifically detect RIP3 in mouse and rat. Human RIP3 can be detected by hRIP3 antibody (8963).
Swissprot #:
Q9QZL0
User NOte:
Optimal dilutions for each application to be determined by the researcher.
VALIDATION:

KO Validation (Figure 7,9,13) shows RIP3 expression detected by anti-RIP3 antibodies (2283) was disrupted in multiple tissues of RIP3 KO mice (figure 7, 13) and in RIP3 KO MEF cells (figure 9).

KD validation (Figure 10): Anti-RIP3 antibody (2283) specificity was further verified by RIP3 specific knockdown. RIP3 signal in L929 cells transfected with RIP3 shRNA was disrupted in comparison with that in cells transfected with control shRNA.

Regulated expression validation (Figure 7,11,13): RIP3 expression detected by anit-RIP3 antibodies (2283) was up-regulated by treatment of cerulein (figure 7), LPS (figure 11) or acetaminophen (figure 13) in different tissues or cells.

Overexpression validation (Figure 12): RIP3 overexpression detected by anit-RIP3 antibodies (2283) was observed in three cell lines stably transfected with RIP3.

References

  1. He et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell. 2009;137(6):1100-11. PMID: 19524512
  2. Zhang et al. Functional complementation between FADD and RIP1 in embryos and lymphocytes. Nature. 2011;471(7338):373-6. PMID: 21368761
  3. Narayan et al. The NAD-dependent deacetylase SIRT2 is required for programmed necrosis. Nature. 2012;492(7428):199-204. PMID: ? 23201684
  4. Li et al. Tissue damage negatively regulates LPS-induced macrophage necroptosis. Cell Death Differ. 2016;23(9):1428-47. doi:10.1038/cdd.2016.21. Epub 2016. PMID: ?????? 26943325
  5. Yang et al. Regulation of RIP3 by the transcription factor Sp1 and the epigenetic regulator UHRF1 modulates cancer cell necroptosis. Cell Death Dis. 2017;8(10):e3084. doi:10.1038/cddis.2017.483. PMID: 28981102
  6. Ramachandran et al. Receptor interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis in mice. Hepatology. 2013;58(6):2099-108. PMID: 23744808
  7. Li et al. The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell. 2012 ;150(2):339-50. PMID: ?????? 22817896
  8. Vitner et al. RIPK3 as a potential therapeutic target for Gaucher's disease. Nat Med. 2014;20(2):204-8. PMID: 24441827
  9. Wang et al. RNA viruses promote activation of the NLRP3 inflammasome through a RIP1-RIP3-DRP1 signaling pathway. Nat Immunol. 2014;15(12):1126-33. PMID: 25326752
  10. Onizawa et al. The ubiquitin-modifying enzyme A20 restricts ubiquitination of the kinase RIPK3 and protects cells from necroptosis. Nat Immunol. 2015;16(6):618-27. PMID: 25939025
  11. Murphy et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity. 2013;39(3):443-53. PMID: 24012422
  12. Nogusa et al. RIPK3 Activates Parallel Pathways of MLKL-Driven Necroptosis and FADD-Mediated Apoptosis to Protect against Influenza A Virus. Cell Host Microbe 2016;20(1):13-24. PMID: 27321907
  13. Pearson et al. EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation. Nat Microbiol. 2017 ;2:16258. PMID: 28085133
  14. Yang et al. The end of RIPK1-RIPK3-MLKL-mediated necroptosis in acetaminophen-induced hepatotoxicity? Hepatology. 2016;64(1):311-2PMID: 26418225
  15. Qu et al. Graphene oxide induces toll-like receptor 4 (TLR4)-dependent necrosisin macrophages. ACS Nano. 2013;7(7):5732-45.PMID: 23734789