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<strong>Figure 1 Western Blot Validation in Human Heart Tissue Lysate</strong><br>Loading: 15 μg of lysates per lane.Antibodies: Smac 2409 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.(A) Without blocking peptide(B) With blocking peptide
<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 2409 (1 μg/mL), 1h incubation at RT in 5% NFM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 4 Western Blot Validation in Mouse 3T3/NIH Cells</strong><br>Loading: 15 μg of lysate.Antibodies: Smac 2409 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 5 Immunohistochemistry Validation of Smac in Human Ovary Tissue </strong><br> Immunohistochemical analysis of paraffin-embedded Human Ovary tissue using anti-Smac antibody (2409) at 5 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 fromSTS-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.
Figure 1 Western Blot Validation in Human Heart Tissue Lysate
Loading: 15 μg of lysates per lane.Antibodies: Smac 2409 (1 μg/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.(A) Without blocking peptide(B) With blocking peptide
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Smac Antibody

CATALOG NUMBER: 2409

Clonality:
Polyclonal
Tested Applications:
ELISA, IF, IHC-P, IP, WB
Host Species:
Rabbit
Species Reactivity:
Human, Mouse, Rat
Conjugate:
Unconjugated
Specifications
Host Species:
Rabbit
Species Reactivity:
Human, Mouse, Rat
Immunogen:
Anti-Smac antibody (2409) was raised against a peptide corresponding to 15 amino acids near the carboxy terminus of human Smac.
The immunogen is located within the last 50 amino acids of Smac.
Conjugate:
Unconjugated
Tested Applications:
ELISA, IF, IHC-P, IP, WB
Application Note:
WB: 1 μg/mL; IHC: 5 μg/mL.
Antibody validated: Western Blot in human, mouse and rat samples; Immunohistochemistry in human samples. All other applications and species not yet tested.
Positive Control 1:
Cat. No. 1301 - Human Heart Tissue Lysate
Positive Control 2:
Cat. No. 1316 - Human Ovary Tissue Lysate
Positive Control 3:
Cat. No. 11-201 - Human Ovary Tissue Slide
Predicted Molecular Weight:
Predicted: 27kD
Observed: 20 kD
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.

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). 2409 can detect human, mouse and rat Smac.
Purification:
Smac Antibody is DEAE purified.
Clonality:
Polyclonal
Isotype:
IgG
physical-state:
Liquid
Buffer:
Smac Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/mL
Storage Conditions:
Smac antibody can be stored at 4°C for three months and -20°C, stable for up to one year. As with all antibodies care should be taken to avoid repeated freeze thaw cycles. Antibodies should not be exposed to prolonged high temperatures.
Ncbi Official Symbol:
Diablo
Additional Names:
Smac Antibody: Smac, AU040403, 0610041G12Rik, 1700006L01Rik, Smac, Diablo homolog, mitochondrial, Direct IAP-binding protein with low pI
Protein Accession Number:
NP_063940
Protein Gi Number:
8953908
Ncbi Gene Id Number:
66593
User Note:
Optimal dilutions for each application to be determined by the researcher.
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 Reference 1:
Du et al. Cell 2000; 102:33-42.
Background Reference 2:
Verhagen et al. Cell 2000;102:43-53.
Background Reference 3:
Srinivasula et al. J. Biol. Chem. 2000; 275:36152-7.
Background Reference 4:
Chai et al. Nature 2000; 406:855-62.
Citation 1:
Ho et al. XIAP activity dictates Apaf-1 dependency for caspase 9 activation. Mol Cell Biol. 2007;27(16):5673-85. PMID: 17562856
Citation 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
Citation 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
Citation 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
Citation 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
Citation 6:
Dohi et al. Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis. J Clin Invest. 2004;114(8):1117-27. PMID: 15489959
Citation 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
Citation 8:
Xia et al. Tumor cell dependence on Ran-GTP-directed mitosis. Cancer Res. 2008;68(6):1826-33. PMID: 18339863
Citation 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
Citation 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
Citation 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
Citation 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
Citation 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
Citation 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
Citation 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
Citation 16:
Ndubaku et al. Antagonism of c-IAP and XIAP proteins is required for efficient induction of cell death by small-molecule IAP antagonists. ACS Chem Biol. 2009;4(7):557-66.PMID: 19492850
Citation 17:
Stichel et al. Mono- and double-mutant mouse models of Parkinson's disease display severe mitochondrial damage. Hum Mol Genet. 2007;16(20):2377-93. PMID: 17412759
Citation 18:
Sun et al. Proteasome inhibitor-induced apoptosis is mediated by positive feedback amplification of PKCdelta proteolytic activation and mitochondrial translocation. J Cell Mol Med. 2008;12(6A):2467-81. PMID: 18298651
Citation 19:
Martin-Latil et al. Bax is activated during rotavirus-induced apoptosis through the mitochondrial pathway. J Virol. 2007;81(9):4457-64. PMID: 17301139
Citation 20:
Mayorga et al. Bcl-2 is a key factor for cardiac fibroblast resistance to programmed cell death. J Biol Chem. 2004;279(33):34882-9. PMID: 15184368

FOR RESEARCH USE ONLY

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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.

CATALOG NUMBER:

2409

List Size:
0.02 mg, 0.1 mg

List Price:

Price range: $99.00 through $445.00

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