Western blot analysis was performed as described previously [48 (link)] using the following antibodies: cIAP1 (R&D Systems, Inc., Wiesbaden, Germany), RIP1 (BD Biosciences, Heidelberg, Germany), RIP3 (Novus Biologicals, Littleton, CO, USA), MLKL (GeneTex, Irvine, CA, USA), phospho-MLKL (Cell Signaling Technologies, Danvers, MA, USA), β-Actin (Sigma-Aldrich) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (HyTest, Turku, Finland) as loading controls and secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany). Alternatively, secondary antibodies labeled with IRDye infrared dyes were used for fluorescence detection (Odyssey Imaging System, LI-COR Bioscience, Bad Homburg, Germany). All Western blots shown are representative of at least two independent experiments.
Ciap1
Manufactured by R&D Systems
Sourced in United States
CIAP1 is a laboratory product offered by R&D Systems. It is a recombinant protein that represents the Baculovirus IAP Repeat (BIR) domain of the human Cellular Inhibitor of Apoptosis Protein 1 (CIAP1). The BIR domain of CIAP1 is responsible for the inhibition of apoptosis or programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (5 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
Ciap2, by Abcam (2 mentions)
Caspase 8, by Enzo Life Sciences (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Mcl 1, by Cell Signaling Technology (2 mentions)
Survivin, by R&D Systems (2 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Irdye 680rd, by LI COR (2 mentions)
Ciap2, by Cell Signaling Technology (2 mentions)
C myc, by Cell Signaling Technology (2 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by HyTest (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Phospho mlkl, by Cell Signaling Technology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Ha sc 805, by Santa Cruz Biotechnology (1 mentions)
Ubiquitin bml pw8810, by Enzo Life Sciences (1 mentions)
17 protocols using ciap1
1
Western Blot Analysis of Cell Signaling
2
Western Blot Antibodies and Reagents
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The following antibodies were used for western blot: HAX1 (BD Transduction Laboratories: 610825, 1:500), cIAP2 (ABCAM: ab32059, 1:1000), cIAP1 (R&D Systems: AF8171, 1:500), FLAG (F1804, 1:5000), His (H1029, 1:3000), and α-tubulin (T6074, 1:5000) (Sigma-Aldrich Co. LLC.), p100/p52 (#3017, 1:1000), NIK (#4994, 1:1000), and cIAP2 (#3130, 1:1000) (Cells Signaling Technology, Inc.), GFP (sc-9996, 1:1000) and HA (sc-805, 1:1000) (Santa Cruz Biotechnology, Inc.), and ubiquitin (BML-PW8810, 1:100, Enzo Life Science). Recombinant human CD40 ligand/TNFSF5 (617-CL) and recombinant human LIGHT/TNFSF14 (664-LI) were obtained from R&D Systems. Pierce anti-HA agarose (26181) was obtained from Thermo Scientific. Anti-FLAG M2 affinity gels (A2220), 3× FLAG peptide (F4799), anti-c-Myc agarose affinity gels (A7470), cycloheximide (C4859), and MG132 (C2211) were purchased from Sigma-Aldrich Co. LLC. Bortezomib (velcade) (MG-341) and protein G plus/protein A agarose suspension (IP05) were purchased from Merck Millipore. Complete protease inhibitor cocktail (13760700) was purchased from Roche.
3
Apoptosis Signaling Pathway Profiling
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The cells or tissue biopsy samples were homogenized in a protein lysis buffer. A 50-μg protein sample was separated through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking, the membranes were immunoblotted with various primary antibodies, followed by incubation with the appropriate peroxidase-coupled secondary antibodies. Furthermore, the signal was visualized on an X-ray film after detecting with an enhanced chemiluminescent detection system. Antibodies to p21, p-p53 (phosphorylated at S15, S46, and S392), tumor necrosis factor receptor I (TNF RI), Fas-associated death domain protein (FADD), cellular inhibitor of apoptosis protein 1 (cIAP-1), hypoxia-inducible factor (HIF)-1α, and TNF-related apoptosis-inducing ligand receptor 1 (TRAIL R1) were purchased from R&D Systems.
4
Protein Extraction and Western Analysis
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Total protein was extracted from cell cultures using RIPA buffer (sc-24948) according
to manufacturer protocol (Santa Cruz Biotechnology, Dallas, TX, USA) and concentrations
estimated with the BCA Protein Assay Kit (Thermo Scientific). SDS-PAGE and western
analysis were performed using, respectively, the NuPage system (Invitrogen) and the
Supersignal Chemiluminescent Substrate system (Thermo Scientific). The following
antibodies were used: cIAP1 (AF8181, R&D Systems), Caspase8 (90A992, Thermo
Scientific and 9746, Cell Signaling Technology, Boston, MA, USA), cFLIP (ab8421, Abcam,
Cambridge, UK), RIP1 (3493, Cell Signaling Technology) and cleaved PARP (9541, Cell
Signaling Technology), MLKL (sc-130172, Santa Cruz Biotechnology), GAPDH (MAB374,
Millipore, Billerica, MA, USA) and β-tubulin (T5201, Sigma-Aldrich).
to manufacturer protocol (Santa Cruz Biotechnology, Dallas, TX, USA) and concentrations
estimated with the BCA Protein Assay Kit (Thermo Scientific). SDS-PAGE and western
analysis were performed using, respectively, the NuPage system (Invitrogen) and the
Supersignal Chemiluminescent Substrate system (Thermo Scientific). The following
antibodies were used: cIAP1 (AF8181, R&D Systems), Caspase8 (90A992, Thermo
Scientific and 9746, Cell Signaling Technology, Boston, MA, USA), cFLIP (ab8421, Abcam,
Cambridge, UK), RIP1 (3493, Cell Signaling Technology) and cleaved PARP (9541, Cell
Signaling Technology), MLKL (sc-130172, Santa Cruz Biotechnology), GAPDH (MAB374,
Millipore, Billerica, MA, USA) and β-tubulin (T5201, Sigma-Aldrich).
5
Immunoblot Analysis of Cell Signaling
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Whole-cell lysates were obtained and analyzed by immunoblot as previously described.39 (link) The following primary antibodies were used: cIAP-1 (1:2000) from R&D Systems; anti-cIAP-2 (1:1000) and anti-IL-6 (ab6672; 1:1000) from Abcam; actin (sc-1615; 1:1000), IκBα (sc-371; 1:1000), NIK (sc-7211; 1:1000) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); RIP1 (BD 610458; 1:1000) from BD Biosciences (San Jose, CA, USA); NF-κB2 p100/p52 (#4882; 1:1000) and PARP (#9532; 1:1000) from Cell Signaling Technology (Beverly, MA, USA); GAPDH (MAB374; 1:5000) from EMD Millipore (Temecula, CA, USA).
6
Molecular Signaling in Cancer Cells
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Antibodies targeting pan-RAS, Noxa (CalBiochem), Actin and ERK1/2 (Sigma), cleaved-PARP, cleaved caspase-3, phosphoERK1/2 (Thr202/Tyr204), pAKT and AKT (Cell Signaling), cIAP1 (R&D Systems), cIAP2 and XIAP (BD Biosciences), caspase-8 (Enzo Life Sciences) and Mcl-1 (Santa Cruz Biotechnology) were employed in western blot experiments. z-VAD(OMe)-FMK was purchased by BIOMOL, Necrostatin-1 from Enzo Life Sciences. PD98059 and UO126 were purchased from CalBiochem, LY294002 from Sigma, GDC-0941 and Triciribine from Selleckem. Infliximab (Schering-Plough) and Enbrel (Wyeth Pharmaceuticals) were used as TNF blockers. CPT and neocarzinostatin were purchased from Sigma-Aldrich, etoposide by Teva. SM83 synthesis has been described elsewhere [25 (link), 28 (link)], while izTRAIL was purified as already shown [29 (link)]. Mutant KRAS (G13D) was cloned in the pINDUCER20 and lentiviral particles prepared modifying an already described protocol [30 (link)] and using Lipofectamine 2000 as transfection reagent. Expression of the transgene was induced by doxycycline (Sigma-Aldrich) at the indicated concentrations.
7
Evaluating Smac Mimetics Effects on IAPs
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RA- and OA-FLS were treated with 20 μM Smac 060 or 066 for 18 h. Whole cell extracts were then prepared by directly lysing the cells in lysis buffer. Cell lysates were processed and the protein concentration was measured with the BCA method (Thermo Scientific, USA), according to the manufacturer’s instructions. Proteins in the cell lysates were separated with SDS–PAGE on 4–12 % Tris–HCl precast gels (Invitrogen, Carlsbad, CA). Proteins in the gel were transferred onto nitrocellulose membranes (Invitrogen, Carlsbad, CA). The membranes were blocked for 3 h with 5 % non-fat dry milk (Lab Scientific) in 0.1 % PBS–Tween 20. Then, membranes were incubated with a primary antibody overnight at 4 °C. Next, we added secondary antibodies conjugated to horseradish peroxidase (Thermo Scientific, USA), and membranes were developed with Western Lightning Plus ECL (PerkinElmer, OH, USA). Densitometry was performed with ImageJ software (National Institutes of Health, Bethesda, USA). We used primary antibodies to the following proteins: cIAP1 (R&D Systems, Minneapolis), cIAP2 (BD Pharmingen, MA, USA), and XIAP (Cell Signalling Technology).
8
Apoptosis Pathway Protein Analysis
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Western blot analyses were performed as described previously [25 ] using the following antibodies: caspase-8, cFLIP, caspase-9, XIAP, Bim, caspase-3, PTEN, p-PTEN (Ser380/Thr382/383), PARP, p-AKT (Ser-473), AKT, β-actin, MCL-1 (all from Cell Signaling, Beverly, MA), cIAP-2 (Epitomics, Burlingame, CA, USA), cIAP-1, survivin (both from R&D Systems), Noxa, and cFLIP (Santa Cruz Biotechnology).
9
Western Blot Analysis of Apoptosis Regulators
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Total cell lysates and western blotting were performed for survivin (R&D Systems, Minneapolis, MN, USA), XIAP (R&D Systems), c-IAP1 (R&D Systems), IκBα (Cell Signaling, Danvers, MA, USA), Akt (Cell Signaling), Phospho-Akt Ser473 (Cell Signaling) and Yb-1 (Abcam, San Francisco, CA, USA) as previously described.8 (link) The subcellular fractionation analysis of NF-κB (Cell Signaling) and Yb-1 was performed according to the manufacturer's instructions (NE-PER Nuclear and Cytoplasmatic Extraction Reagent Kit; Thermo Scientific, Waltham, MA, USA). To assess Pgp expression (monoclonal anti-Pgp clone C219, 1:10.000), cell lysates were prepared as previously described.19 (link) Total protein was loaded onto 3–8% gradient NuPAGE Novex Tris-acetate gels (Invitrogen), and proteins were transferred to Hybond-P membranes (GE Healthcare, Buckinghamshire, UK). We normalized the total protein to β-actin (Sigma?Aldrich Corp., St. Louis, MO, USA) and Na+K+ATPase (Cell Signaling) and the subcellular fraction to lamina B (Calbiochem - Darmstadt, Germany) and HSC70 (Santa Cruz, Dallas, TX, USA).
To visualize protein expression, we used the ECL detection system according to the manufacturer's instructions (GE Healthcare).
To visualize protein expression, we used the ECL detection system according to the manufacturer's instructions (GE Healthcare).
10
Western Blot Protein Detection
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Cell lysates were prepared with radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) supplemented with Complete protease inhibitor cocktail and PhosSTOP – Phosphatase Inhibitor Cocktail (Roche). Standard procedures for Western blotting were followed using the following primary antibodies: cIAP-1 (AF8181; R&D), XIAP (610762; BD Biosciences) and β-actin (A1978; Sigma-Aldrich). Specific protein bands were visualized using IRDye 680RD or 800CW secondary antibodies (LI-COR) and LI-COR Odyssey infrared imaging system.
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