Western blotting analysis was performed as previously described [30 (link)]. Anti-cIAP1 was obtained from R&D System (Minneapolis, MN). Anti-Bax and cIAP2 antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA). Anti-Bak and xIAP antibodies were obtained from Cell Signaling Biotech (Danvers, MA), anti-Bcl-2, and Bcl-xL antibodies were obtained from BD Biosciences (San Diego, CA), and anti-β-actin was obtained from Sigma (St Louis, MO).
Anti ciap1
Manufactured by R&D Systems
Sourced in United States
Anti-cIAP1 is a laboratory reagent that specifically binds and neutralizes the cellular Inhibitor of Apoptosis Protein 1 (cIAP1). cIAP1 is an important regulator of cell survival and apoptosis. Anti-cIAP1 can be used in research applications to study the role of cIAP1 in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (3 mentions)
Anti xiap, by Cell Signaling Technology (3 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Anti xiap, by BD (2 mentions)
Anti cleaved caspase 8, by R&D Systems (2 mentions)
Anti p100 p52, by Cell Signaling Technology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Anti lamin a c 4c11, by Cell Signaling Technology (1 mentions)
Mg132, by Peptide Institute (1 mentions)
Anti phospho iκbα ser32 36 5a5, by Cell Signaling Technology (1 mentions)
Anti iκbα c 21, by Santa Cruz Biotechnology (1 mentions)
Anti ikkα h 744, by Santa Cruz Biotechnology (1 mentions)
Anti nik, by Cell Signaling Technology (1 mentions)
Anti relb c 19, by Santa Cruz Biotechnology (1 mentions)
Anti phospho p65, by Cell Signaling Technology (1 mentions)
Donkey anti mouse igg, by LI COR (1 mentions)
Anti p52, by Santa Cruz Biotechnology (1 mentions)
Donkey anti goat igg, by LI COR (1 mentions)
Donkey anti rabbit igg, by LI COR (1 mentions)
Anti p65, by Santa Cruz Biotechnology (1 mentions)
10 protocols using anti ciap1
1
Western Blotting Analysis of Apoptosis Regulators
2
Immunoblotting Analysis of NF-κB Pathway
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Whole-cell (30 µg), cytoplasmic (30 µg) and nuclear (10 µg) lysates were resolved by SDS-PAGE and analyzed by immunoblotting. The following antibodies were used: anti-NF-κB2 p52 (C-5) (Santa Cruz Biotechnology, #sc-7386) for detection of p52 and its precursor p100; anti-NIK (Cell Signaling, Danvers, MA, USA, #4994); anti-RelB (C-19) (Santa Cruz Biotechnology, #sc-226); anti-phospho-p100 (Ser866/870) (Cell Signaling, #4810); anti-phospho-IκBα (Ser32/36) (5A5) (Cell Signaling, #5205); anti-IκBα (C-21) (Santa Cruz Biotechnology, #sc-371); anti-phospho-IKKα(Ser180)/IKKβ (Ser181) (Cell Signaling, #2681), anti-IKKα (H-744, Santa Cruz Biotechnology, #sc-7218), anti-IKKα/β (H-470, Santa Cruz Biotechnology, #sc-7607), anti-TRAF2 (C90-481) (BD Biosciences, San Jose, CA, USA, 558890); anti-TRAF3 (H-122) (Santa Cruz Biotechnology, SC-1828); anti-cIAP1 (R&D systems, Minneapolis, MN, USA, AF8181); anti-lamin A/C (4C11) (Cell signaling, #4774); anti-α-tubulin (Sigma-Aldrich, St Louis, MO, USA, T9026). For detection of endogenous NIK protein, cells were treated with 0.1% DMSO or 20 µM of MG132 (PEPTIDE INSTITUTE, Osaka, Japan) for 6 hours and cytoplasmic extracts were subjected to immunoblot analysis.
3
Antibodies for Western Blot Analysis
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Western blot analysis was performed as described previously [17] (link) using the following antibodies: anti-cIAP1 (R&D Systems, Inc., Wiesbaden-Nordenstadt, Germany), anti-cIAP2 (Epitomics, Burlingam, CA), anti-XIAP from BD Biosciences, anti–phospho-p65 from Cell Signaling (Beverly, MA), anti-phospho-IκBα and anti-IκBα (Cell Signaling), anti-β-actin (Sigma), anti-p65, and anti-p52 from Santa Cruz Biotechnology (Santa Cruz, CA). Donkey anti-mouse IgG, donkey anti-rabbit IgG, or donkey anti-goat IgG labeled with IRDye infrared dyes was used for fluorescence detection at 680 or 800 nm (LI-COR Biotechnology, Bad Homburg, Germany).
4
Ceramide Analogs Induced Apoptosis Analysis
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Western blotting analysis was performed as previously described69 (link). Briefly, tumor cells were cultured in the presence of the indicated ceramide analogs or ceramide analogs plus MegaFasL for 4 h. Cells were collected and lysed in cytosol buffer [10 mM Hepes, pH 7.4, 250 mM Sucrose, 70 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, protease and phosphatase inhibitor cocktails (Calbiochem, Billerica, MA), and 0.01% digitonin] for 10 min. Cytosolic fractions were resolved in 4–20% SDS polyacrylamide gel and analyzed by Western blotting. Anti-cleaved caspase 8 and anti-cIAP1 were obtained from R&D Systems. Anti-cleaved human PARP, anti-xIAP, anti-p38, anti-p-p38, anti-pErk1/2, anti-Erk1/2, anti-pJNK, and anti-JNK antibodies obtained from Cell Signaling. β-actin was obtained from Sigma-Aldrich.
5
Antibody Validation in Cell Culture
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N-acetylcysteine was obtained from Sigma-Aldrich (St. Louis, MO, USA, A9165). The antibodies were obtained as follows: anti-actin (Chemicon, Billerica, MA, USA, 1501); anti-AKT (Cell Signaling, Danvers, MA, USA, 9272); anti-Bad (Santa Cruz, Dallas, TX, USA, sc-7869); anti-Bax (Santa Cruz, sc-493); anti-Bcl-2 (Santa Cruz; sc-509); anti-Bcl-XL (Cell Signaling, 2764); anti-Bim (Cell Signaling, 2933); anti-Caspase 2 (Cell Signaling, 2224); anti-Caspase 3 (Imgenex, San Diego, CA, USA, IMG-144A); anti-cIAP1 (R&D Systems, Minneapolis, MN, USA, AF8181); anti-GAPDH (Santa Cruz, sc-32233); anti-ISCU (Santa Cruz; sc-373694); anti-Mcl-1 (Santa Cruz, sc-819); anti-pAKT (S472/473) (Cell Signaling, 4058); anti-Puma (Cell Signaling, 4976); anti-XIAP (Cell Signaling, 2045).
6
Comprehensive Western Blot Analysis of Cell Death Signaling
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Western blot was performed as described [21 (link)]. Primary antibodies used were anti-actin (1:2000 MP Biomedicals C4), anti-caspase-8 (1:300 Cell Signaling #9746 and 1:300, Abcam ab32125), anti-caspase-3 (1:1000 Cell Signaling #9662), anti-caspase-7 (1:400 Cell Signaling #12827), anti-RIP1 (1:500 BD Biosciences 610458), anti-XIAP (1:400 BD Biosciences 610762), anti-cIAP1 (1:200 R&D Systems AF8181), anti-p100/p52 (1:500 Cell Signaling #4882) and anti-c-FLIP (1:400 Cell Signaling #5634). Secondary horseradish peroxidase-labeled antibodies were from GE Healthcare and Dako and were used at 1:5000 dilutions. For the chemiluminescent reaction, Supersignal Substrate (Thermo Scientific) was used. Chemiluminescence was detected with a LAS-1000 CCD camera and Image Reader LAS-1000 Pro v2.6 software (Fujifilm, Tokyo, Japan) or an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).
7
Co-immunoprecipitation Assay Protocol
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Anti‐EndoG (Proteintech, Rosemont, IL, USA), antiactin (Bethyl, Montgomery, TX, USA), anti‐HA (Covance, Emeryville, CA, USA), anti‐PARP‐1 (Santa Cruz Biotechnology, Inc. Dallas, TX, USA), anti‐His, anti‐GFP, anti‐β‐tubulin, anti‐myc 9E10 (Millipore, Billerica, MA, USA), and anti‐cIAP1 (R&D Systems, Minneapolis , MN, USA) were used for WB or co‐ IP assays.
Co‐IP was performed as follows unless otherwise noted. Cells were lysed in lysis buffer (50 mm Tris pH 8.0, 150 mm NaCl, 1 mm EDTA, 10% glycerol, 1% Triton X‐100, protease inhibitor cocktail). WCLs were mixed with the indicated antibody for 2 h at 4 °C. Protein‐A sepharose beads (Sigma‐Aldrich) were incubated with the immunocomplex for 2 h at 4 °C and then washed three times with IP wash buffer (20 mm Tris pH 8.0, 150 mm NaCl, 1 mm EDTA, 1% Triton X‐100). Samples were subjected to sodium dodecyl‐sulfate polyacrylamide gel electrophoresis and analyzed by WB.
Co‐IP was performed as follows unless otherwise noted. Cells were lysed in lysis buffer (50 m
8
Western Blot Analysis of Apoptosis Regulators
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Western blotting analysis was performed as previously described [65 (link)]. Anti-cleaved caspase 8 (Cat#: AF705, 0.5μg/ml) and anti-cIAP1 (Cat#: AF8181, 1:500) were obtained from R&D systems. Anti-caspase 3 (Cat#: 9661, 1:1000), anti-caspase 9 (Cat#: 9501, 1:500), anti-PARP (Cat#: 9541, 1:250), anti-xIAP (Cat#: 2042, 1:500), anti-p100/p52 (Cat#: 4882, 1:1000), anti-IκBα (Cat#: 4814, 1:2000), anti-pIκBα (Cat#: 2859, 1:1000) antibodies were obtained from Cell Signaling. Anti-cIAP2 (Cat#: NB110-57030, 1:250) was obtained from Novus. Anti-β-actin (Cat#: A5441, 1:5000) was obtained from Sigma (St Louis, MO).
9
Investigating NF-κB Pathway Regulation
10
E2F1 and cIAP1 Immunoprecipitation
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Cells (106 per conditions) were transfected with plasmids encoding E2F1 wt or K161/164 R mutant, cIAP1 wt or DP1. Forty-eight hours after transfection, cells were washed with PBS and lysed in IP Lysis buffer (150 mM de NaCl, 50 mM Tris HCl pH 7,4, 20 mM EDTA, 0,5% NP40, 1 mM DTT, 5 mM N-ethylmaleimide (Sigma-Aldrich) and protease inhibitors) for 30 min at 4 °C. Lysate supernatants were precleaned by using 20 μl of Protein A/G+ Agarose beads (Sigma) and incubated (overnight, 4 °C) with rabbit polyclonal anti-E2F1 (C20) antibody (Santa Cruz Biotechnology), mouse IgG1 purified anti-HA.11 (Biolegend), goat polyclonal anti-cIAP1 (R&D Systems) or Igg anti-mouse or anti-rabbit antibody. Beads were then washed in IP lysis buffer and denaturated in Laemmli buffer before immunoblot analysis.
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