Antibodies directed against A20 (5630; Cell Signaling), ABIN-1 (HPA037893; Sigma Aldrich), c-IAP1 (ALX-803-335; Enzo and 4952; Cell Signaling), caspase 8 (9496, 4790; Cell Signaling and 804–429, Enzo), CC8 (8592, Cell Signaling), RIPK1 (3493; Cell Signaling), caspase 3 (9662; Cell Signaling), CC3 (9661; Cell Signaling), PARP (9532 and 9542; Cell Signaling), cFLIP (56343; Cell Signaling), mouse RIPK3 (95702; Cell Signaling), phospho-RIPK3 (T231/S232; ab205421; Abcam), phospho-RIPK1 (Ser166) rodent specific (31122; Cell Signaling), IKKβ (8943; Cell Signaling), phospho-IKKα/β (2697; Cell Signaling), and GAPDH (MAB374; Millipore) were used for Western blot as described below. Recombinant mouse TNF-α was purchased from R&D. Recombinant TWEAK (R&D) and TRAIL (Peprotech), were used at a final concentration of 250 ng/ml. FasL (R&D) was used at a final concentration of 40 ng/ml with anti-HA at 2.5 μg/ml (BioLegend). Nec1s (2263; BioVision) and emricasan (HY-10396; MedChemExpress) were used at 50 μM final concentration.
C flip
Manufactured by Cell Signaling Technology
Sourced in United States
C-FLIP is a lab equipment product from Cell Signaling Technology. It is a protein involved in the regulation of programmed cell death, or apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Bcl xl, by Cell Signaling Technology (6 mentions)
Mcl 1, by Cell Signaling Technology (6 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (5 mentions)
Cleaved caspase 3, by Cell Signaling Technology (5 mentions)
Caspase 3, by Cell Signaling Technology (4 mentions)
Bcl 2, by Cell Signaling Technology (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Caspase 8, by Cell Signaling Technology (4 mentions)
Caspase 9, by Cell Signaling Technology (3 mentions)
Ripk1, by Cell Signaling Technology (2 mentions)
Grp78, by Cell Signaling Technology (2 mentions)
P jnk, by Cell Signaling Technology (2 mentions)
α tubulin, by Cell Signaling Technology (2 mentions)
Ciap2, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Cleaved caspase 8, by Cell Signaling Technology (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
20 protocols using c flip
1
Antibodies for Apoptosis Signaling
2
Comprehensive Signaling Pathways Analysis
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Antibodies to BAX, BAK, BCL-XL, CHOP, c-FLIP, IRE1, RIP1, iNOS, FADD, Cathepisin B, mTOR, phospho-mTOR S2448 and S2481, phospho-RAPTOR S792, TSC2 T1426, PTEN, phospho-PTEN S380, ATF6, eNOS, AIF, XBP1, NOXA, PUMA, ATG5 phospho-ATG13 S318, Beclin-1, AKT, phospho-AKT T308, eiF2α, phospho-eiF2α S51, phospho p65 S536, ATF4, PGKI and II, TRX, SOD, ATM, phospho-ATM S1981, AMPKα, phospho-AMPK T172, phospho-ULK1 S757, S317, STAT3, STAT5, p70 S6K, phospho-ERK1/2, GRP78, HSP70 and HSP90, phospho-γH2AX, were purchased from Cell Signaling Technology, (Danvers, MA). PERK, CD95 and caspase 9 antibodies, were purchased from Santa Cruz Biotechnology, (Dallas, TX).
3
Vanillin Oxime Induces Apoptosis
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The A549 and NCI-H2170 cells were exposed to 12.5 and 15 µM concentrations of vanillin oxime for 48 h. After exposure, incubation was carried with ice-cold (0.5 ml) lysate buffer (consisting of NaCl [5 M], NaVO4 [0.2 M], Nonidet P-40 [10%], EGTA [0.1 M], EDTA [0.5 M], PhCH2SO2F [0.1 M], NaF [1 M], HEPES [1 M] aprotinin [2 µg/ml] and leupeptin [2 µg/ml]) for 45 min. After centrifugation at 12,000 ×g at 4°C protein level in supernatant separated from the lysates was estimated by Bio-Rad assay (Bio-Rad, Hercules, CA, USA). Fractionation of 60 µg protein samples on SDS-PAGE was followed by transfer to PVDF membranes (Millipore, Bedford, MA, USA). Probing of proteins was carried out by over-night membrane incubation at 4°C with primary antibodies. Washing membranes with TBST was followed by incubating the blots for 1 h with anti-rabbit secondary antibodies conjugated to horseradish peroxidase. Detection as well as analysis of the bands was carried out using the ECL western blot analysing system (Biotech Inc., USA). The primary antibodies used were against JNK, p-JNK, caspase-3, -8, -9, CHOP, Bcl-xL, ERK1/2, p-ERK1/2, DR4, DR5, survivin, Bax, cFLIP, xIAP (Cell Signaling Technology, Danvers, MA, USA).
4
Apoptosis Pathway Protein Detection
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Antibody to caspase-3 was obtained from Imgenex (San Diego, CA, USA). Antibodies to caspase-9, c-FLIP, p-JNK, JNK, GRP78, and CHOP were purchased from Cell Signaling (Beverley, MA, USA). Antibody to p-eIF2α was from StressGen (Ann Arbor, MI, USA). Antibody to caspase-8 was obtained from Calbiochem (San Diego, CA, USA). All other antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DMEM and fetal bovine serum were obtained from Gibco-BRL (Grand Island, NY, USA). Polyvinylidene difluoride (PVDF, 0.22 mm) membrane was purchased from Bio-Rad (Hercules, CA, USA). [g-32P] ATP was from BMS (Avenue, NY, USA). All the other reagents were obtained from Sigma (St. Louis, MO, USA).
5
Thyroid Cancer Cell Line Signaling Pathways
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Reagents included the following: lexatumumab (Human Genome Sciences, GlaxoSmithKline, Philadelphia, PA, USA), LY294002 (Cell Signaling Technology, Danvers, MA, USA) and PLX4720 (Plexxikon, Berkeley, CA, USA). Antibodies – β-actin, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (phospho-ERK), p44/42 MAPK (ERK1/2) (Total ERK), total Akt, phospho-Akt (Ser473), caspases 3,8,9, cleaved caspase-3, PARP, c-FLIP, TRAIL-R2, Bcl2, Bcl-xL, Mcl-1, Bim, Bid, Bax, Bad, Bak were purchased from Cell Signaling Technology.
ATC cell lines – 8505c (BRAFV600E/−), SW1736 (BRAFV600E/wt), HTh-7 (NRASQ61R); PTC cell lines – BCPAP (BRAFV600E/wt), TPC-1 (RET/PTC-1); and normal thyroid cell line – HTori were grown in RPMI1640 medium with 10% FBS and Penicillin (100 units/ml)/streptomycin (100 μg/ml) (Gibco, Grand Island, NY, USA).
ATC cell lines – 8505c (BRAFV600E/−), SW1736 (BRAFV600E/wt), HTh-7 (NRASQ61R); PTC cell lines – BCPAP (BRAFV600E/wt), TPC-1 (RET/PTC-1); and normal thyroid cell line – HTori were grown in RPMI1640 medium with 10% FBS and Penicillin (100 units/ml)/streptomycin (100 μg/ml) (Gibco, Grand Island, NY, USA).
6
Immunoblotting Analysis of Apoptosis Signaling
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Cells were collected and lysed for protein extraction and the followed immunoblotting as previously described [12 (link)]. Phosphorylation and level of protein was demonstrated by using antibodies against human cellular proteins, including caspase-3, caspase-9, caspase-8, cytochrome c, Poly (ADP-ribose) polymerase (PARP), Bcl-2, Bak, Bax, Fas, FasL, phosphorylated P38 (p-P38), total P38, phosphorylated JNK (p-JNK), total JNK, phosphorylated glycogen synthase kinase 3beta (GSK3β) (Ser9), cellular FLICE inhibitory protein (c-FLIP), and α-tubulin (Cell signaling, Beverly, MA). Detection of antigen–antibody complex was performed by using ECL reagent (Millipore, Bedford, MA, USA) and luminescence image system (LAS-4000 mini; Fujifilm, Tokyo, Japan). Semi-quantitation of reacted signals was determined using Multi Gauge software version 2.2 (FujiFilm, Tokyo, Japan). Three independent analyses were performed for statistical analysis.
7
Molecular Mechanisms of Cell Death
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The following inhibitors and drugs were used at the indicated concentration unless otherwise specified in the Figure or Figure legends: dinaciclib 50 nM (Selleck-Chemicals, Houston, TX, USA, cat# S2768), NVP-2 50 nM (Selleck-Chemicals Cat# S8981), etoposide (Absource diagnostic, Munich, Germany, cat# S1225-0100), cisplatin (Selleck-Chemicals, cat# S1166), Propidium Iodide (Sigma-Aldrich, Cat# P4864). Recombinant iz-huTRAIL was provided by H. Walczak. Antibodies against the following antigens were used: RNA pol II RBP1 pSer2 (Biolegend, San Diego, CA, USA, cat# 920204), RNA pol II (Biolegend, cat# 904001), MCL-1 (Cell Signalling, Danvers, MA, USA, cat# 5453), cFLIP (Cell Signalling, cat# 56343), C-Myc (Cell Signalling, cat# 5605), PARP (BD, Franklin Lakes, NJ, USA, cat# 556362), caspase 9 (Abcam, Cambridge, UK, cat# 202068), caspase 8 (Cell Signalling, cat# 9746), cleaved caspase 3 (Cell Signalling, cat# 9664), β-Actin (Sigma-Aldrich, cat# A1978), Tubulin (Sigma-Aldrich, cat# T9026), Rabbit IgG (SouthernBiotech, Birmingham, AL, USA, cat# 4050-05), Mouse IgG (SouthernBiotech, cat# 1031-05). VC-1 was produced and provided by Vichem Chemie Research Ltd.
8
LMB100 Cytotoxicity Mechanism Evaluation
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LMB100 (RG7787, huSS1(Fab)-LR-GGS-LO10-PE24) was supplied by Roche Innovation Center (Penzberg, Germany). Panbinostat was purchased from Selleck (Houston, TX). Antibodies detecting EF2, BAX, BAK, BID, BCLxl, MCL1, Caspase- 9 and cleaved caspase-3 and −8, PARP, CIAP2, c-FLIP, BNIP3L and GAPDH were purchased from Cell Signaling (Danvers, MA).
9
Regulation of TRAIL and NF-κB in BM-MDSCs
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For the SB conditioned medium studies, BM-MDSCs were plated in 10-cm dishes, and on the following day were incubated with an equal volume of SB cell conditioned medium for 0, 0.5, 1, 2, or 4 hours. BM-MDSC cytoplasmic and nuclear protein lysates were collected using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (#78833; Thermo Fisher) per the manufacturer’s protocol. Proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The following primary antibodies were used for immunoblotting analysis: polyclonal TRAIL (ab231265 or ab42121-100; Abcam), IκBα (#4814s; Cell Signaling Technology), phospho-IκBα (Ser32) (#2859s; Cell Signaling Technology), phospho-IκBα (Ser36) (#ab133462; Abcam), NF-κB p65 (#SC-8008; Santa Cruz), Lamin B2 (#12255; Cell Signaling Technology), histone H3 (#4499s; Cell Signaling Technology), and cFLIP (#5634s; Cell Signaling Technology). Membranes were blotted with primary antibody overnight at 4°C at a dilution of 1:1000. Goat anti-mouse or rabbit IgG (H + L)–horseradish peroxidase (HRP) conjugate secondary antibodies were used at a dilution of 1:2000. Proteins were visualized with Clarity and Clarity Max ECL Western Blotting Substrates (Bio-Rad).
10
Apoptosis Induction Pathway Analysis
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Bortezomib (BTZ) and THZ1 (Selleck Chemicals, Houston, TX, USA) were dissolved in dimethyl sulfoxide (DMSO) (Sigma, MO, USA) and stored at −80 °C, Human TNF-α (PeproTech, CA, USA). The antibodies for Mcl-1 (# SC-12,756 Santacruz Biotech. USA), Bak (#3814), Bcl-xL (#2764), Survivin (#2808), PARP(#9532), Cleaved caspase 3 (#9661), c-FLIP (#56,343), NF-kB pathway antibody sampler kit (#9936), CyclinD1(#2978), CyclinD2 (#3741), pRB 807(#9307) were from Cell Signalling Technology (Cell Signalling Tech. MA, USA), P53 (# SC-126, Santacruz Biotech, USA), CDK2 (# RPB889Hu03, Cloud-clone corp. USA) and β-Actin (Cell Signaling Technology, USA) were used.
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