ProSci

Smac Antibody

Product Code:
 
PSI-2411
Product Group:
 
Primary Antibodies
Supplier:
 
ProSci
Host Type:
 
Rabbit
Antibody Isotype:
 
IgG
Antibody Clonality:
 
Polyclonal
Regulatory Status:
 
RUO
Applications:
  • Enzyme-Linked Immunosorbent Assay (ELISA)
  • Immunofluorescence (IF)
  • Immunohistochemistry (IHC)
  • Western Blot (WB)
1 / 10
<strong>Figure 1 Western Blot Validation in (A and B) Mouse Heart Tissue Lysate and (C) Rat Heart Tissue Lysate </strong><br> Loading: 15 μg of lysates per lane. Antibodies: Smac 2411 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. A Mouse heart B Mouse heart and blocking peptide C Rat heart
2 / 10
<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: Smac 2409 (1 μg/mL), Smac 2411 (1 μg/mL), and beta-actin (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
3 / 10
<strong>Figure 3 Western Blot Validation in Human, Mouse and Rat Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: Smac 2411 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
4 / 10
<strong>Figure 4 Immunofluorescence Validation of Smac in Mouse Spleen Cells</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed Mouse Spleen Cells labeling Smac with 2411 at 10 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
5 / 10
<strong>Figure 5 Immunohistochemistry Validation of Smac in Mouse Spleen Tissue </strong><br> Immunohistochemical analysis of paraffin-embedded Mouse Spleen Tissue using anti-Smac antibody (2411) at 2 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 / 10
<strong>Figure 6 KO Validation in Mouse Fibroblasts and Myoblasts (Ho et al., 2007)</strong><br> The indicated MEFs or MEMs were exposed to 2 μM STS for 4 h and analyzed by Western blot. Accumulation of Smac/Diablo in mitochondrion-depleted cytosol fractions from STS-treated Apaf-1 KO cells were detected by anti-smac antibodies. Smac expression was not detected in smac KO mice.
7 / 10
<strong>Figure 7 Immunohistochemistry Validation of Smac in Human gastric carcinoma (Kim et al., 2011) </strong><br> Smac was highly expressed in gastric mucosa of patients with gastric carcinoma.
8 / 10
<strong>Figure 8 Immunofluorescence Analysis of Smac in NB4-LR1 Cells (Saumet et al., 2005) </strong><br> NB4-LR1 cells were either treated with ATRA(1 μM) for 3 days without or with the T3C1 recombinant fragment (3 μM) or treated with staurosporine (STP; 5 μM ) for 3.5 hours. STP, but not ATRA or AYRA/T3C1 induced the release of smac.
9 / 10
<strong>Figure 9 Induced Expression Validation in Rat Liver (Genestier et al., 2005)</strong><br> Mitochondria from rat liver were treated with increasing concentrations of rPVL (A), rLukS (B), or Bax alpha (C) for 1 hour at 30 ˚C. rPVL induces the release of the apoptogenic proteins cytochrome c and Smac/DIABLO from isolated mitochondria.
10 / 10
<strong>Figure 10 Overxpression Validation in HEK293T Cells (Flygare et al., 2012)</strong><br> HEK293T cells were transiently transfected with Smac and Myc-tagged cIAP1, cIAP2, ML-IAP, or empty vector. Cells were lysed, and lysates were incubated with the indicated concentrations of 1 and immunoprecipitated with anti-Myc antibody (left panels). Samples were then immunoblotted with anti-Smac and anti-Myc antibodies. Whole-cell lysates are shown in the right panel.
❮❯

<strong>Figure 1 Western Blot Validation in (A and B) Mouse Heart Tissue Lysate and (C) Rat Heart Tissue Lysate </strong><br> Loading: 15 μg of lysates per lane. Antibodies: Smac 2411 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. A Mouse heart B Mouse heart and blocking peptide C Rat heart
<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: Smac 2409 (1 μg/mL), Smac 2411 (1 μg/mL), and beta-actin (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 3 Western Blot Validation in Human, Mouse and Rat Cell Lines</strong><br> Loading: 15 μg of lysates per lane. Antibodies: Smac 2411 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 4 Immunofluorescence Validation of Smac in Mouse Spleen Cells</strong><br> Immunofluorescent analysis of 4% paraformaldehyde-fixed Mouse Spleen Cells labeling Smac with 2411 at 10 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
<strong>Figure 5 Immunohistochemistry Validation of Smac in Mouse Spleen Tissue </strong><br> Immunohistochemical analysis of paraffin-embedded Mouse Spleen Tissue using anti-Smac antibody (2411) at 2 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 KO Validation in Mouse Fibroblasts and Myoblasts (Ho et al., 2007)</strong><br> The indicated MEFs or MEMs were exposed to 2 μM STS for 4 h and analyzed by Western blot. Accumulation of Smac/Diablo in mitochondrion-depleted cytosol fractions from STS-treated Apaf-1 KO cells were detected by anti-smac antibodies. Smac expression was not detected in smac KO mice.
<strong>Figure 7 Immunohistochemistry Validation of Smac in Human gastric carcinoma (Kim et al., 2011) </strong><br> Smac was highly expressed in gastric mucosa of patients with gastric carcinoma.
<strong>Figure 8 Immunofluorescence Analysis of Smac in NB4-LR1 Cells (Saumet et al., 2005) </strong><br> NB4-LR1 cells were either treated with ATRA(1 μM) for 3 days without or with the T3C1 recombinant fragment (3 μM) or treated with staurosporine (STP; 5 μM ) for 3.5 hours. STP, but not ATRA or AYRA/T3C1 induced the release of smac.
<strong>Figure 9 Induced Expression Validation in Rat Liver (Genestier et al., 2005)</strong><br> Mitochondria from rat liver were treated with increasing concentrations of rPVL (A), rLukS (B), or Bax alpha (C) for 1 hour at 30 ˚C. rPVL induces the release of the apoptogenic proteins cytochrome c and Smac/DIABLO from isolated mitochondria.
<strong>Figure 10 Overxpression Validation in HEK293T Cells (Flygare et al., 2012)</strong><br> HEK293T cells were transiently transfected with Smac and Myc-tagged cIAP1, cIAP2, ML-IAP, or empty vector. Cells were lysed, and lysates were incubated with the indicated concentrations of 1 and immunoprecipitated with anti-Myc antibody (left panels). Samples were then immunoblotted with anti-Smac and anti-Myc antibodies. Whole-cell lysates are shown in the right panel.

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Further Information

Additional Names:
Smac Antibody: Smac, AU040403, 0610041G12Rik, 1700006L01Rik, Smac, Diablo homolog, mitochondrial, Direct IAP-binding protein with low pI
Application Note:
WB: 1 μg/mL; IF: 10 μg/mL; IHC: 2 μg/mL.

Antibody validated: Western Blot in mouse and rat samples; Immunofluorescence and Immunohistochemistry in mouse samples. All other applications and species not yet tested.
Background:
Smac Antibody: The inhibitor of apoptosis proteins (IAPs) regulate programmed cell death by inhibiting members of the caspase family of enzymes. A novel mammalian protein that binds to IAPs and neutralizes the inhibitory effect of IAPs on caspases was recently identified and designated Smac/DIABLO. Smac/DIABLO is a mitochondrial protein that is released along with cytochrome c during apoptosis and activates cytochrome c/Apaf-1/capase-9 pathway. Analysis of the structural basis of Smac/DIABLO reveals that the N-terminal amino acids are required for binding of Smac/DIABLO to IAPs and activation of caspases. Smac/DIABLO is expressed in a variety of human and mouse tissues.
Background References:
  • Du et al. Cell 2000; 102:33-42.
  • Verhagen et al. Cell 2000;102:43-53.
  • Srinivasula et al. J. Biol. Chem. 2000; 275:36152-7.
  • Chai et al. Nature 2000; 406:855-62.
Buffer:
Smac 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-Smac antibody (2411) was raised against a peptide corresponding to 16 amino acids near the carboxy terminus of murine Smac.

The immunogen is located within the last 50 amino acids of Smac.
ISOFORMS:
Human Smac has 3 isoforms, including isoform 1 (239aa, 27.1kD), isoform 2 (186aa, 21.2kD), and isoform 3 (195aa, 22.3kD). Mouse Smac has one isoform (237aa, 26.8kD) and Rat Smac also has one isoform (237aa, 26.9kD). 2411 can detect human, mouse and rat.
NCBI Gene ID #:
66593
NCBI Official Name:
diablo homolog (Drosophila)
NCBI Official Symbol:
Diablo
NCBI Organism:
Mus musculus
Physical State:
Liquid
PREDICTED MOLECULAR WEIGHT:
Predicted: 27kD

Observed: 20 kD
Protein Accession #:
AF203914
Protein GI Number:
8953908
Purification:
Smac Antibody is DEAE purified.
Research Area:
Apoptosis,Cancer
Swissprot #:
Q9JIQ3
User NOte:
Optimal dilutions for each application to be determined by the researcher.
VALIDATION:

Independent Antibody Validation in Cell lines (Figure 2) shows similar Smac expression profile in human and mouse cell lines detected by two independent anti-Smac antibodies that recognize different epitopes, 2409 against human C-terminus domain and 2411 against murine C-terminus domain.  Smac proteins are detected in the most tested cell lines at different expression levels by the two independent antibodies.

KO Validation (Figure 6): Anti-Smac antibody specificity was further verified by Smac knockout mice. Smac signal smac KO mice was disrupted as compared to control.

Induced Expression Validation (Figure 6,9): Smac expression detected by anit-Smac antibodies was up-regulated by STS or rPVL treatment.

Overexpression Validation (Figure 10): Smac overexpression was detected by anit-Smac antibodies on smac transfected HEK293T cells.

References

  1. Ho et al. XIAP activity dictates Apaf-1 dependency for caspase 9 activation. Mol Cell Biol. 2007;27(16):5673-85. PMID: 17562856
  2. Kim et al. Expression of apoptosis-related proteins and its clinical implication in surgically resected gastric carcinoma. Virchows Arch. 2011;459(5):503-10. PMID: 21947931
  3. Saumet et al. Type 3 repeat/C-terminal domain of thrombospondin-1 triggers caspase-independent cell death through CD47/alphavbeta3 in promyelocytic leukemia NB4 cells. Blood. 2005;106(2):658-67. PMID: 15784731
  4. Genestier et al. Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils. J Clin Invest. 2005;115(11):3117-27. PMID: 16276417
  5. Flygare et al. Discovery of a potent small-molecule antagonist of inhibitor of apoptosis (IAP) proteins and clinical candidate for the treatment of cancer(GDC-0152). J Med Chem. 2012;55(9):4101-13. PMID: 22413863
  6. Dohi et al. Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis. J Clin Invest. 2004;114(8):1117-27. PMID: 15489959
  7. Watanabe et al. MITOPLD is a mitochondrial protein essential for nuage formation and piRNA biogenesis in the mouse germline. Dev Cell. 2011;20(3):364-75. PMID: 21397847
  8. Xia et al. Tumor cell dependence on Ran-GTP-directed mitosis. Cancer Res. 2008;68(6):1826-33. PMID: 18339863
  9. Otera et al. Drp1-dependent mitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling. J Cell Biol. 2016;212(5):531-44.PMID: 26903540
  10. Lucken-Ardjomande et al. Contributions to Bax insertion and oligomerization of lipids of the mitochondrial outer membrane. Cell Death Differ. 2008;15(5):929-37. PMID: 18259190
  11. Lucken-Ardjomande et al. Bax activation and stress-induced apoptosis delayed by the accumulation of cholesterol in mitochondrial membranes. Cell Death Differ. 2008;15(3):484-93. PMID: 18084240
  12. Sedelies et al. Blocking granule-mediated death by primary human NK cells requires both protection of mitochondria and inhibition of caspase activity. Cell Death Differ. 2008;15(4):708-17. PMID: 18202705
  13. Waterhouse et al. Functional dissociation of DeltaPsim and cytochrome c release defines the contribution of mitochondria upstream of caspase activation during granzyme B-induced apoptosis. Cell Death Differ. 2006;13(4):607-18. PMID: 16167065
  14. Becatti et al. The involvement of Smac/DIABLO, p53, NF-kB, and MAPK pathways in apoptosis of keratinocytes from perilesional vitiligo skin: Protective effects of curcumin and capsaicin. Antioxid Redox Signal. 2010;13(9):1309-21. PMID: 20085492
  15. Moubarak et al. FAIM-L is an IAP-binding protein that inhibits XIAP ubiquitinylation and protects from Fas-induced apoptosis. J Neurosci. 2013;33(49):19262-75. PMID: 24305822