Smac Antibody Cat. No.: 2409

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

Figure 2 Independent Antibody Validation (IAV) via Expression Profile in Cell Lines Figure 3 Western Blot Validation in Human, Mouse and Loading: 15 μg of lysates per lane. Antibodies: Smac 2409 Rat Cell Lines (1 μg/mL), Smac 2411 (1 μg/mL), and beta-actin (1 μg/mL), Loading: 15 μg of lysates per lane. Antibodies: Smac 2409 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat (1 μg/mL), 1h incubation at RT in 5% NFM/TBST. anti-rabbit IgG HRP conjugate at 1:10000 dilution. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.

Figure 5 Immunohistochemistry Validation of Smac in Human Ovary Tissue Immunohistochemical analysis of paraffin-embedded Figure 4 Western Blot Validation in Mouse 3T3/NIH Human Ovary tissue using anti-Smac antibody (2409) at 5 Cells μg/ml. Tissue was fixed with formaldehyde and blocked Loading: 15 μg of lysate. Antibodies: Smac 2409 (1 μg/mL), with 10% serum for 1 h at RT; antigen retrieval was by 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat heat mediation with a citrate buffer (pH6). Samples were anti-rabbit IgG HRP conjugate at 1:10000 dilution. 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.

October 2, 2021 1 https://www.prosci-inc.com/smac-antibody-2409.html Figure 6 KO Validation in Mouse Fibroblasts and Myoblasts (Ho et al., 2007) Figure 7 Immunohistochemistry Validation of Smac in The indicated MEFs or MEMs were exposed to 2 μM STS Human gastric carcinoma (Kim et al., 2011) for 4 h and analyzed by Western blot. Accumulation of Smac was highly expressed in gastric mucosa of patients Smac/Diablo in -depleted fractions with gastric carcinoma. from STS-treated Apaf-1 KO cells were detected by anti- smac antibodies. Smac expression was not detected in smac KO mice.

Figure 9 Induced Expression Validation in Rat Liver (Genestier et al., 2005) Figure 8 Immunofluorescence Analysis of Smac in NB4- Mitochondria from rat liver were treated with increasing LR1 Cells (Saumet et al., 2005) concentrations of rPVL (A), rLukS (B), or Bax alpha (C) for 1 NB4-LR1 cells were either treated with ATRA(1 μM) for 3 hour at 30˚C. rPVL induces the release of the apoptogenic days without or with the T3C1 recombinant fragment (3 cytochrome c and Smac/DIABLO from isolated μM) or treated with staurosporine (STP; 5 μM ) for 3.5 mitochondria. hours. STP, but not ATRA or AYRA/T3C1 induced the release of smac.

Figure 10 Overxpression Validation in HEK293T Cells (Flygare et al., 2012) 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.

Specifications

HOST SPECIES: Rabbit

SPECIES REACTIVITY: Human, Mouse, Rat

Anti-Smac antibody (2409) was raised against a peptide corresponding to 15 amino acids near the carboxy terminus of human Smac. IMMUNOGEN:

The immunogen is located within the last 50 amino acids of Smac.

TESTED APPLICATIONS: ELISA, IF, IHC-P, IP, WB

October 2, 2021 2 https://www.prosci-inc.com/smac-antibody-2409.html WB: 1 μg/mL; IHC: 5 μg/mL.

APPLICATIONS: 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

2) Cat. No. 1316 - Human Ovary Tissue Lysate

3) Cat. No. 11-201 - Human Ovary Tissue Slide

Predicted: 27kD PREDICTED MOLECULAR WEIGHT: Observed: 20 kD

Advanced 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. VALIDATION: 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.

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 ISOFORMS: Rat Smac also has one isoform (237aa, 26.9kD). 2409 can detect human, mouse and rat Smac.

Properties

PURIFICATION: Smac Antibody is affinity chromatography purified via peptide column.

CLONALITY: Polyclonal

ISOTYPE: IgG

CONJUGATE: Unconjugated

PHYSICAL STATE: Liquid

BUFFER: Smac Antibody is supplied in PBS containing 0.02% sodium azide.

CONCENTRATION: 1 mg/mL

Smac antibody can be stored at 4˚C for three months and -20˚C, stable for up to one STORAGE CONDITIONS: year. As with all antibodies care should be taken to avoid repeated freeze thaw cycles. Antibodies should not be exposed to prolonged high temperatures.

October 2, 2021 3 https://www.prosci-inc.com/smac-antibody-2409.html Additional Info

OFFICIAL SYMBOL: Diablo

Smac Antibody: Smac, AU040403, 0610041G12Rik, 1700006L01Rik, Smac, Diablo homolog, ALTERNATE NAMES: mitochondrial, Direct IAP-binding protein with low pI

ACCESSION NO.: NP_063940

PROTEIN GI NO.: 8953908

GENE ID: 66593

USER NOTE: Optimal dilutions for each application to be determined by the researcher.

Background and References

Smac Antibody: The inhibitor of proteins (IAPs) regulate by inhibiting members of the family of enzymes. A novel mammalian protein that binds to IAPs and neutralizes the inhibitory effect of IAPs on was recently identified and designated Smac/DIABLO. Smac/DIABLO is a mitochondrial protein that is BACKGROUND: 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.

REFERENCES: 1) Du et al. Cell 2000; 102:33-42.

2) Verhagen et al. Cell 2000;102:43-53.

3) Srinivasula et al. J. Biol. Chem. 2000; 275:36152-7.

4) Chai et al. Nature 2000; 406:855-62.

CITATIONS

1)Ho et al. XIAP activity dictates Apaf-1 dependency for caspase 9 activation. Mol Cell Biol. CITATIONS: 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 (IAP) proteins and clinical candidate for the treatment of cancer(GDC-0152). J Med Chem. 2012;55(9):4101-13. PMID: 22413863

October 2, 2021 4 https://www.prosci-inc.com/smac-antibody-2409.html 6)Dohi et al. Mitochondrial 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

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

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

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

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

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

ANTIBODIES FOR RESEARCH USE ONLY.

For additional information, visit ProSci's Terms & Conditions Page.

October 2, 2021 5 https://www.prosci-inc.com/smac-antibody-2409.html