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Smac diablo

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Smac/DIABLO is a protein that plays a role in the regulation of apoptosis, a process of programmed cell death. It is released from the mitochondria during apoptosis and helps to activate caspases, which are enzymes that execute the cell death program. Smac/DIABLO can inhibit the activity of certain proteins that normally suppress caspase activation, thereby facilitating apoptosis.

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Lab products found in correlation

Gapdh, by Santa Cruz Biotechnology (2 mentions) Bcl2 associated x (bax), by Santa Cruz Biotechnology (2 mentions) Cytochrome c, by Santa Cruz Biotechnology (2 mentions) Phosphorylated jnk, by Cell Signaling Technology (1 mentions) Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions) Alexafluor 488 conjugated anti goat secondary antibody, by Thermo Fisher Scientific (1 mentions) Z vad fmk, by Merck Group (1 mentions) Necrostatin 1 nec 1, by Merck Group (1 mentions) Phosphorylated h2a x, by Cell Signaling Technology (1 mentions) Transfection reagent, by Qiagen (1 mentions) α tubulin, by Santa Cruz Biotechnology (1 mentions) β actin antibody, by Merck Group (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Bcl 2, by Santa Cruz Biotechnology (1 mentions) Rhtrail, by Thermo Fisher Scientific (1 mentions) Mouse anti actin mab, by Merck Group (1 mentions) Enhanced chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) P atf2, by Santa Cruz Biotechnology (1 mentions) Caspase 7, by Santa Cruz Biotechnology (1 mentions)

6 protocols using smac diablo

1

Neuronal Cell Death Pathway Signaling

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Antibodies to XIAP, RIP-1, phosphorylated JNK, and phosphorylated H2AX were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies to actin, GAPDH, AIF, Smac/DIABLO, and calpain were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The calpain inhibitor PD150606 was purchased from Calbiochem (La Jolla, CA, USA), the pancaspase inhibitor z-VAD-fmk and necroptosis inhibitor necrostatin-1 (Nec-1) from Sigma-Aldrich (St. Louis, MO, USA) and the Ambion Silencer Select RIP-1 siRNA from Life Technologies. It was transfected into neuronally differentiated PC12 cells using Lipofectamine RNAiMAX Reagent (Life Technologies) as per manufacturer’s instructions. Alexafluor 488-conjugated anti-goat secondary antibody was purchased from Invitrogen (Carlsbad, CA, USA) and horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies from Cell Signaling Technologies.
Bollino D., Balan I, & Aurelian L. (2015). Valproic acid induces neuronal cell death through a novel calpain-dependent necroptosis pathway. Journal of neurochemistry, 133(2), 174-186.
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2

Apoptosis Induction by 23-HUA

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23-HUA was isolated from C. bancoensis and its structure was confirmed spectroscopically (1H-NMR, 13C-NMR, IR and MS) as reported previously [18 (link)] The purity of the isolated 23-HUA was determined to be >97% (HPLC). MTT, β-actin antibody, protein A, DAPI, PI and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Primary antibodies targeting cytochrome c, caspase-8, FasL and FADD were obtained from BD Biosciences Pharmingen (San Jose, CA, USA). Antibodies for caspase-3, caspase-9, Bid, tBid, Bcl-2, Bax, PARP, Fas, Smac/DIABLO, α-tubulin, COX 4 and secondary antibody were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Caspase inhibitors (z-VAD-fmk, z-DEVD-fmk, z-IETD-fmk, and z-LEHD-fmk) were purchased from Calbiochem (San Diego, CA, USA). siRNA for caspase-8, Fas, FADD and transfection reagents were purchased from Qiagen GmbH (Hilden, Germany).
Won J.H., Chung K.S., Park E.Y., Lee J.H., Choi J.H., Tapondjou L.A., Park H.J., Nomura M., Hassan A.H, & Lee K.T. (2018). 23-Hydroxyursolic Acid Isolated from the Stem Bark of Cussonia bancoensis Induces Apoptosis through Fas/Caspase-8-Dependent Pathway in HL-60 Human Promyelocytic Leukemia Cells. Molecules, 23(12), 3306.
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3

MART-1 and Cytochrome C Antibodies Protocol

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Antibodies specific to MART-1 and anti-cytochrome C and smac/DIABLO were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and DAKO (Carpinteria, CA), respectively. Mouse anti-actin mAb was obtained from Chemicon. rhTRAIL was purchased from Peprotech (NJ). DR5, Bcl-xL and c-IAP-2 siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). DR5 expression plasmid and Drozitumab were kindly provided by Dr. Avi Ashkenazi (Genentech Inc., San Fransisco, CA) under Material Transfer Agreement (MTA). Blocking Abs and fluorochrome conjugated Abs for FACS analyses were purchased from eBiosciences (San Diego, CA). Suberoylanilide hydroxanic acid (SAHA) procured commercially, was diluted in dimethyl sulfoxide (DMSO). DMSO concentration did not exceed 0.1% in any experiment.
Jazirehi A.R., Kurdistani S.K, & Economou J.S. (2014). Histone Deacetylase Inhibitor Sensitizes Apoptosis-resistant Melanomas to Cytotoxic Human T Lymphocytes through Regulation of TRAIL/DR5 Pathway. Journal of immunology (Baltimore, Md. : 1950), 192(8), 3981-3989.
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4

Immunoblot Analysis of Apoptosis-Related Proteins

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Total proteins in Hep-2 and Hep-2/R cells were extracted by using RIPA lysis buffer on ice for 30 min. 50 μg total proteins were separated with SDS-PAGE before transferring to a nitrocellulose membrane. The membrane was then blocked with skimmed milk and incubated with primary antibody of ATF2, p-ATF2, c-Jun, Bcl-xl, cytochrome c, Smac/DIABLO, caspase-9, caspase-7, caspase-3 and GAPDH (dilution 1:200, Santa Cruze, USA) at 4 °C overnight. Subsequently, the membrane were labeled with HRP-conjugated secondary antibodies (dilution 1:5000, Santa Cruze, USA) for 2 h at room temperature. Proteins on the membrane were probed by using an enhanced chemiluminescent substrate (Thermo Fisher Scientific, Inc, USA).
Tian L., Zhang J., Ren X., Liu X., Gao W., Zhang C., Sun Y, & Liu M. (2017). Overexpression of miR-26b decreases the cisplatin-resistance in laryngeal cancer by targeting ATF2. Oncotarget, 8(45), 79023-79033.
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5

Medicarpin Synthesis and Cell Assays

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Medicarpin (Med), a naturally occurring phytoalexin was synthesized in gram scale at the medicinal process chemistry division of the CSIR-Central Drug Research Institute, India as per a standardized procedure.45 (link) The Med stock (20 mM in DMSO, stored at −20°C) solution was diluted in cell culture media for experimental use. The Super killer TRAIL/Apo2L and the antagonistic antibodies against DR4 (HS101) and DR5 (HS201) were purchased from Alexis Biosciences (San Diego, CA, USA). The primary antibodies against DR4, c-FLIP, Bid, tBid, Cytochrome C, Smac/Diablo, and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), while the antibodies against DR5, Survivin, CHOP, XIAP, JNK, p-JNK, Bip, eIF2α, p-eIF2α, cleaved caspase-8, cleaved caspase-9, cleaved caspase-3, and cleaved caspase-7 were purchased from Cell Signaling Technology (Boston, MA, USA). Primary antibodies against Bcl-2 and Bax and the 7AAD/Annexin-based cell death assay kit were purchased from BD Biosciences (San Jose, CA, USA). All the other biochemicals were from Sigma (St Louis, MO, USA) unless otherwise stated.
Trivedi R., Maurya R, & Mishra D.P. (2014). Medicarpin, a legume phytoalexin sensitizes myeloid leukemia cells to TRAIL-induced apoptosis through the induction of DR5 and activation of the ROS-JNK-CHOP pathway. Cell Death & Disease, 5(10), e1465-.
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6

Immunofluorescence Localization of Endogenous Proteins

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For standard immunofluorescence (IF) detection of endogenous
proteins, cells were plated in 8-well microscopy μ-slides (Ibidi),
transfected and treated as indicated, and fixed in 4% PFA for 15 min.
Following, cells were permeabilized with 0.3% Triton X-100 in PBS for 10 min
and blocked with 3% BSA for 45 min. Cells were then incubated with primary
antibodies against BAX (Santa Cruz Biotechnology; ABclonal), cytochrome
c (BD Biosciences), Rabex-5 (Santa Cruz Biotechnology),
Smac/DIABLO (Santa Cruz Biotechnology), TOM20 (Santa Cruz Biotechnology),
TOM70 (Santa Cruz Biotechnology), at room temperature for 1 hr, or with Rab5
(Cell Signaling) or ALS2 (Sigma-Aldrich; ABclonal) antibodies at 4°C
overnight. Fluorescence staining was performed for 30 min at room
temperature using highly cross-adsorbed Alexa Fluor 488, 546 or 647
secondary antibodies (Thermo Fisher Scientific). Following primary and
secondary antibody incubations, cells were washed three times in PBS. To
visualize nuclei, cells were stained with 1 μg/mL Hoechst 33342 in
PBS for 10 min.
Wang T.S., Coppens I., Saorin A., Brady N.R, & Hamacher-Brady A. (2020). Endolysosomal Targeting of Mitochondria is Integral to BAX-mediated Mitochondrial Permeabilization During Apoptosis Signaling. Developmental cell, 53(6), 627-645.e7.
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