THP-1 cells were treated with SEB and lysed, and their total protein content was determined as described in Caspase Activity Assay Section. Approximately 25 μg of protein was separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidine difluoride (PVDF) membrane (Mini Trans-Blot Cell, BioRad). The PVDF membrane was blocked with 5% (m/v) non-fat milk (Boster, Wuhan, China) in TBST buffer (50 mM Tris, 138 mM NaCl, 2.7 mM KCl, pH 8.0, with 0.05% (v/v) Tween 20; ZSGB-BIO, Beijing, China) for 1 h and incubated in anti-HLA-DRa (1:200; Santa Cruz Biotechnology, Paso Robles, CA, USA), anti-TNFR1 (1:200; Santa Cruz), and anti-β-actin (1:1000, Boster) diluted in 5% (m/v) non-fat milk at 4°C overnight. The membrane was washed five times with TBST, incubated in goat-anti-mouse secondary antibody conjugated with horseradish peroxidase (HRP; 1:3000, Boster) diluted in 5% (m/v) non-fat milk for 1 h, and washed five times with TBST. Immunoreactivity was visualized by using a SuperSignal West Dura substrate (Thermo Fisher Scientific). Bands were detected by using ChemiDoc Touch Imaging System (BioRad) and densitometrically analyzed with ImageJ 1.51c (Wayne Rasband, National Institutes of Health, USA).
Anti tnfr1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-TNFR1 is a laboratory product that targets the tumor necrosis factor receptor 1 (TNFR1). It is used for research purposes in cellular and molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti bcl 2, by Cell Signaling Technology (2 mentions)
Anti caspase 8, by Cell Signaling Technology (2 mentions)
Anti iκbα, by Santa Cruz Biotechnology (2 mentions)
Anti tnfr2, by Santa Cruz Biotechnology (2 mentions)
Anti phospho jnk1 2, by Cell Signaling Technology (2 mentions)
Anti traf2, by Santa Cruz Biotechnology (2 mentions)
Anti jnk2, by Santa Cruz Biotechnology (2 mentions)
Supersignal west dura substrate, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Boster Bio (1 mentions)
Nonfat milk, by Boster Bio (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
60 mm culture dish, by BD (1 mentions)
Anti c parp, by Cell Signaling Technology (1 mentions)
Anti tnfr2, by Cell Signaling Technology (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
13 protocols using anti tnfr1
1
Western Blot Analysis of THP-1 Cell Proteins
2
Western Blotting Analysis of Protein Expression
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The protein expression levels were evaluated by Western blotting. Briefly, 1 × 106 cells in 60 mm culture dish (BD Falcon) were incubated with the indicated concentrations of SAHA for 24 hours. Then cells were washed with PBS and added in 4 volumes of lysis buffer (Intron Biotechnology, Seongnam, Gyeonggi-do, Korea). Thirty μg of total protein were resolved by 4–20% SDS-PAGE gels, and then transferred to Immobilon-P PVDF membranes (Millipore) by electroblotting. Then membranes were probed with anti-PARP, anti-c-PARP, anti-Bax, anti-Bcl-2, anti-TNFR2 (Cell signaling Technology, Danvers, MA), anti-TNFR1, anti-β-actin and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Blots were developed using an EZ-Western Lumi Pico ECL solution kit (DoGen, Seoul, Korea).
3
Antibody Validation for Cell Signaling
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Anti-EphA4, anti-EphA7, and anti-TNFR1 antibodies were purchased from Santa Cruz Biotechnology (USA). Anti-caspase-8 and anti-cleaved caspase-3 antibodies were purchased from Cell Signaling Technology. FITC-conjugated goat anti-human IgG, rhodamine-conjugated anti-rabbit IgG, and FITC-conjugated anti-rabbit IgG antibodies were purchased from Invitrogen (USA). Horseradish peroxidase-conjugated anti-rat IgG and anti-rabbit IgG antibodies were acquired from Zymed (USA).
4
Isoproterenol and Trichostatin A Modulate Cell Signaling
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Isoproterenol and Trichostatin A (TSA) were purchased from Sigma Aldrich (St. Louis, MO, USA) and used at 10 µM and 100 nM, respectively. Murine TNF-α was a gift from the VIB Department for Molecular Biomedical Research of Ghent University (VIB-UGent, Gent, Belgium) and was used at 2000 IU/ml. Insulin-like growth factor-1 (IGF-1) was from ImmunoTools (Friesoythe, Germany) and was used at 10 ng/ml. Anti-β2-AR, anti-TNF-R1, anti-myogenin, anti-PARP, anti-P-H3-Ser10, anti-CBP, anti-RNA polymerase II, anti-p65, anti-IκBα, anti-P-CREB-Ser133 and anti-PKAc were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-P-p65-Ser536, anti-P-ERK-Thr202/Tyr204, anti-P-JNK-Thr183/Tyr185, anti-P-p38-Thr180/Tyr182, anti-P-MSK-1-Thr581, anti-Lamin A/C and anti-CREB were from Cell Signaling Technology (Danvers, MA, USA). Anti-Ac-H3-Lys27 and GAPDH were from AbCam (Cambridge, UK). Anti-α-tubulin and anti-α-actin were from Sigma-Aldrich. In figures, the expression “antibody anti-” was substituted by the Greek letter “α-”. AatII and HincII restriction enzymes were obtained from New England BioLabs (Ipswich, MA, USA).
5
Antibodies for Neurological Biomarkers
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Anti-MMP-12, anti-MMP-9, anti-GFAP, anti-MOG, anti-Iba1, anti-TNFα, anti-TNFR1, anti-TNFR2, anti-caspase-3, anti-cytochrome C, anti-AIF, and anti-MBP antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-NeuN antibody was obtained from Millipore (Billerica, MA). Anti-glial fibrillary acidic protein (GFAP) was obtained from Dakocytomation (Carpinteria, CA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Novus Biologicals (Littleton, CO).
6
Elucidating TNF-α Signaling Pathways
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Minimal essential medium-alpha (α-MEM), fetal bovine serum (FBS), and TRIzol were purchased from Invitrogen (Carlsbad, CA). Hybond C membrane and ECL Western blotting detection system were from Amersham Biosciences (Buckinghamshire, UK). Recombinant human TNF-α and the anti-TNFR1 neutralizing antibody were from R&D System (Minneapolis, MN). Luciferase assay kit was from Promega (Madison, WI). Metafectene transfection reagent was from Biontex Lab (GmbH, Planegg/Martinsried, Germany). SMARTpool RNA duplexes corresponding to Src, TRAF2, ERK2, p38 MAPK, JNK2, and scrambled #2 siRNA were from Dharmacon Research Inc (Lafayette, CO). Anti-phospho-IKKα/β, anti-phospho-NF-κB p65 (Ser536), anti-phospho-c-Src, anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, and anti-phospho-IκB-α antibodies were from Cell Signaling (Danver, MA). anti-NF-κB (p65), anti-lamin A, anti-TRAF2, anti-TNFR1, anti-c-Src, anti-ERK2, anti-p38, anti-JNK2, anti-IκB-α, and anti-sICAM-1 antibodies were from Santa Cruz (Santa Cruz, CA). The anti-GAPDH antibody was from Biogenesis (Boumemouth, UK). Actinomycin D (Act.D), cycloheximide (CHI), PP1, U0126, SB202190, SP600125, GM6001, MMP2/9 inhibitor, and Bay11-7082 were from Biomol (Plymouth Meeting, PA). Enzymes and other chemicals were from Sigma (St. Louis, MO).
7
Immunocytochemistry and Immunoblotting Assays
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Immunocytochemistry was performed with polyclonal rabbit anti-CL5 (Invitrogen catalog #34-1600), goat anti-ICAM1 (R and D Systems, Minneapolis, MN; #BBA17), phalloidin-647 (Invitrogen) and DAPI (Invitrogen). Immunoblotting was performed with mouse mAbs anti-CL5 clone 4C3C2 (Invitrogen catalog #35-2500), anti-β-actin clone AC-74 (Sigma #A2228), anti-HSP90 clone 68 (BD Transduction Labs #610418); anti-myosin light chain 2 clone 19D3.1 (Millipore #MABT180) and anti-TNFR1 (Santa Cruz Biotechnology,#sc-8436); with purified rabbit polyclonal anti-phospho-(Thr18/Ser19) myosin light chain (Cell Signaling #3674); rabbit anti-ROCK1 clone C8F7 (Cell Signaling #4035), rabbit anti-ROCK2 clone D1B1 (Cell Signaling #9029), and rabbit mAb anti-MYPT1 Clone D6C1 (Cell Signaling #8574) and with goat anti-ICAM (R&D #BBA17). Cytokines used were recombinant human tumor necrosis factor-alpha (TNF; Invitrogen catalog #PHC3015) and bovine thrombin (GE Healthcare #27-0846-01). Inhibitors used were Bay11 (Tocris Bioscience #1743) for IκK-β, and Y-27632 (#688000) and H-1152 (#555550; dimethylfasudil) both from EMD Millipore for ROCK.
8
Quantification of Immune Receptors
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Cell protein homogenates (25 μg) were separated by electrophoresis in 12% polyacrylamide gels, transferred to nitrocellulose membranes, and blocked overnight with 5% nonfat dry milk. The membranes were washed and incubated in the presence of the following monoclonal antibodies: anti-IL-1R1, anti-IL-1R2, anti-TNFR1, anti-TNFR2, anti-VDR (sc-393998, sc-376247, sc-8436, sc-393614, and sc-13133, respectively; Santa Cruz Biotechnology, CA, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, MAB374, Millipore, Milford, MA, USA) overnight at 4°C. Membranes were incubated in the presence of secondary antibody conjugated with horseradish peroxidase (sc-2031, Santa Cruz Biotechnology) for 2 hours at room temperature. The immunoblots were visualized by chemiluminescence using ECL Plus (Amersham Pharmacia, UK).
9
Western Blot Analysis of Apoptosis Regulators
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Whole cell lysates were prepared, resolved and blotted as previously described [5 (link)]. Membranes were probed with the following primary antibodies: anti-PTEN (Cell Signaling, Beverly, MA, USA); anti-c-FLIP (Enzo Life Sciences, Exeter, UK); anti-cIAP-1 (Santa Cruz, Heidelberg, Germany); anti-Bcl-2 (Cell Signaling, Beverly, MA, USA); anti-TNFR-1 (Santa Cruz, Heidelberg, Germany); and anti-Caspase-8 (Millipore, Billerica, MA, USA) at 4°C overnight. Membranes were washed three times with 1X PBS + 0.05% Tween-20 before being incubated with the appropriate horseradish peroxidase (HRP)-tagged secondary antibody (GE Healthcare, UK). Immunolabelled proteins were detected using the Luminata Crescendo substrate (Millipore, Billerica, MA, USA). Membranes were reprobed with GAPDH primary antibody (ABD Serotec, UK) to ensure equal loading.
10
Investigating NF-κB Signaling Pathway
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Proteins were resolved using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The membranes were blocked with 5% dry milk in Tris-buffered saline-Tween. Primary antibodies were incubated overnight at 4 °C and diluted according to the manufacturer’s instructions. The following antibodies were used: anti-NF-κB p65 (6956, Cell Signaling, Frankfurt am Main, Germany), anti-phospho-NF-κB p65 (pp65; 3033, Cell Signaling), anti-YB-1 (Eurogentec, Liège, Belgium), anti-TRAF2 (558,890, BD Biosciences), anti-RIP (610,459, BD Biosciences, Heidelberg, Germany), IκBα (9242, Cell Signaling), anti-p-IκBα (9246, Cell Signaling), anti-TNFR1 (8436, Santa Cruz, CA, USA), anti-vinculin (59,803, Santa Cruz), anti-mono- and polyubiquitinated protein (clone FK2, Enzo), anti-cleaved caspase-3 (9661, Cell Signaling), anti-caspase-8 (4790, Cell Signaling), anti-cleaved caspase-8 (9496, Cell Signaling), and anti-pIKKα/β (2697, Cell Signaling). Secondary antibodies coupled to horseradish peroxidase (Southern Biotech, Birmingham, AL, USA) were used for immuno-detection. The detection was performed using Pierce ECL Western blotting substrate (32106, Thermo Fischer Scientific, Waltham, MA, USA).
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