Equal amounts of protein were separated on 10% SDS polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The PVDF membranes were incubated overnight at 4°C with primary antibodies, including COX-2, IκB-α, phosphorylated-IκB-α, lamin B1, p65, phosphorylated-IκB-α (Santa Cruz, CA, USA), ERK1/2, p38, JNK, phosphorylated-ERK1/2, phosphorylated-p38, phosphorylated-JNK (Millipore), ICAM-1, and β-actin (Sigma). After the overnight incubation, the membrane was washed with TBST buffer (150 mM NaCl; 10 mM Tris, pH 8.0; and 0.1% Tween 20) and incubated with HRP-conjugated secondary antibodies for 1 h. Finally, the membranes were washed and processed with Luminol/Enhancer Solution (Millipore) for detection and quantification of the specific protein using the BioSpectrum 600 system (UVP, Upland, CA, USA).
Phosphorylated iκb α
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Phosphorylated-IκB-α is a laboratory protein that has been post-translationally modified by the addition of a phosphate group to the IκB-α protein. IκB-α is a regulatory protein that inhibits the activity of the NF-κB transcription factor. Phosphorylation of IκB-α targets it for degradation, allowing NF-κB to become active and translocate to the nucleus.
Automatically generated - may contain errors
Lab products found in correlation
Biospectrum 600 system, by Analytik Jena (5 mentions)
Luminol enhancer solution, by Merck Group (3 mentions)
β actin, by Merck Group (3 mentions)
Cox 2, by Santa Cruz Biotechnology (3 mentions)
Lamin b1, by Santa Cruz Biotechnology (3 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Phosphorylated erk1 2, by Cell Signaling Technology (2 mentions)
Phosphorylated jnk, by Cell Signaling Technology (2 mentions)
Icam 1, by Merck Group (1 mentions)
Pparα, by Santa Cruz Biotechnology (1 mentions)
Rabbit insulin elisa kit, by Crystal Chem (1 mentions)
Total iκbα, by Santa Cruz Biotechnology (1 mentions)
Crebh, by Santa Cruz Biotechnology (1 mentions)
Antibodies against grp78, by Cell Signaling Technology (1 mentions)
Pi3k and phosphorylated pi3k, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phosphorylated akt, by Santa Cruz Biotechnology (1 mentions)
Las 4000 mini, by Fujifilm (1 mentions)
Anti vcam 1, by Santa Cruz Biotechnology (1 mentions)
Anti icam 1, by Santa Cruz Biotechnology (1 mentions)
11 protocols using phosphorylated iκb α
1
Western Blot Analysis of Inflammatory Proteins
2
Hepatic Metabolic Regulation Protocol
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Chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA) unless indicated otherwise. Antibodies against phosphorylated JNK, total JNK, phosphorylated IκBα, total IκBα, PPARα, and CREBH were purchased from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA, USA). Antibodies against GRP78, IRE1a, Xbp1s, β-actin and the secondary antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Antibodies against FGF21 were purchased from Abcam (Boston, MA, USA). The kit for determining ALT, AST, and FFA were purchased from Abcam. The kit for determining TG, TC, and HDL weaspurchased from Fujifilm Wako Diagnostics USA (Mountain View, CA, USA). The glycogen enzyme-linked immunosorbent assay (ELISA) kit was purchased from BioAssay Systems (Hayward, CA, USA). The rabbit insulin ELISA kit was purchased from Crystal Chem (Elk Grove Village, IL, USA). The periodic acids staining kit, Gomori's trichrome staining kit were purchased from Fisher Scientific (Hampton, NH, USA). The assay kits and antibodies information are listed in Table S2 .
3
Western Blot Analysis of Protein Signaling
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Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with specific antibodies, including IκB-α, lamin B1, Nrf2, HO-1, and phosphorylated-IκB-α (Santa Cruz, CA, USA), phosphorylated-p38, phosphorylated-ERK 1/2, phosphorylated-JNK, and β-actin (Sigma), and p38, ERK1/2, COX-2, JNK, and ICAM-1 (Cell Signaling Technology, MA, USA). Next, the membranes were washed and treated with antibody-HRP conjugates and the protein signals were detected using an enhanced chemiluminescence solution. Images were obtained by a BioSpectrum 600 system (UVP, Upland, CA, USA).
4
Western Blot Analysis of Signaling Proteins
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Proteins were separated on SDS polyacrylamide gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4°C with specific primary antibodies, including Akt, phosphorylated-Akt, COX-2, IκB-α, phosphorylated-IκB-α, p65, and Lamin B1 (Santa Cruz, CA, USA); ERK1/2, phosphorylated-ERK 1/2, p38, phosphorylated-p38, JNK, and phosphorylated-JNK (Millipore); PI3K and phosphorylated-PI3K (Cell Signaling Technology, Inc., MA, USA); and ICAM-1 and β-actin (Sigma). The membrane was washed and incubated with secondary antibodies for 1 h. Finally, specific proteins were detected with Luminol/Enhancer Solution (Millipore) using the BioSpectrum 600 system (UVP, Upland, CA, USA).
5
Protein Extraction and Western Blot Analysis Protocol
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The cells were washed twice with PBS and harvested in a lysis buffer containing 1% SDS, 100 mM NaCl, 50 mM Tris-HCl (pH 8.0) and 20 mM EDTA. Total protein was extracted from the harvested cells using RIPA Buffer (Pierce; Thermo Fisher Scientific, Inc.). Nuclear proteins were extracted using NE-PER kits (Pierce; Thermo Fisher Scientific, Inc.). All proteins were quantified using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). For western blot analysis, ~30 μg protein was electrophoresed on a 10% SDS-PAGE gel and then transferred onto a nitrocellulose membrane. The blots were blocked in 5% non-fat milk in PBS for one hour, and then incubated with rat monoclonal anti-ICAM-1 (1: 500), anti-VCAM-1 (1: 500), anti-RKIP (1: 2,000), rabbit polyclonal anti-p65 (1: 300), anti-inhibitor of NF-kappaBα (IκB-α;1: 250) or phosphorylated IκB-α (1: 1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) antibodies overnight at 37°C. The membrane was then incubated with peroxidase-conjugated secondary antibodies for one hour. Peroxidase activity on the membrane was visualized on x-ray films using a standard enhanced chemiluminescence procedure. The immunoreactive bands were visualized using Immuno Star LD (Wako Pure Chemical, Osaka, Japan), and then measured using LAS-4000 Mini (Fuji Film Co., Ltd., Tokyo, Japan).
6
Western Blot Analysis of Signaling Proteins
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Proteins were extracted with RIPA Lysis Buffer (Thermo, USA) containing proteinase inhibitors (Thermo, USA) and phosphatase inhibitors (Thermo, USA) and then resolved on 10% SDS-PAGE. Western blotting was performed by transferring the proteins onto polyvinylidene difluoride membranes (Millipore, Germany) using a TransBlot system (Bio-Rad, USA). The membranes were washed in ddH2O and then blocked with 5% milk in Tris-buffered saline supplemented with 0.1% Tween 20 (TBST) for 2 h at room temperature. After that, the membranes were incubated overnight at 4°C with antibodies against phosphorylated STAT3 (Cell Signaling, USA), STAT3 (Cell Signaling, USA), phosphorylated STAT4 (Cell Signaling, USA), STAT4 (Cell Signaling, USA), phosphorylated IκBα (Santa Cruz, USA), IκBα (Santa Cruz, USA), phosphorylated p65 (Santa Cruz, USA), p65 (Santa Cruz, USA) or β-actin (Santa Cruz, USA) followed by the incubation with HRP-conjugated secondary antibodies for another 1 h at room temperature. After incubation with the secondary antibodies, signals were detected using West Pico Chemiluminescent Substrate (Pierce, USA) according to the manufacturer's instructions.
7
Fisetin Modulates BEAS-2B Cell Responses
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BEAS-2B cells (2 × 105 cells/mL) were treated with fisetin (0–30 μM) at 37 °C for 1 h, and then stimulated with 10 ng/mL TNF-α at 37 °C for 24 h. BEAS-2B cells were also treated with fisetin and then stimulated with 10 ng/mL TNF-α for 30 min to detect protein phosphorylation. Proteins were extracted from lung tissue and BEAS-2B cells using RIPA buffer solution, and nuclear proteins were extracted using nuclear and cytoplasmic extraction reagents (Thermo Scientific) as described previously [19 (link)]. Next, the proteins were separated on SDS acrylamide gels by electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked in 5% nonfat skim milk and incubated with specific primary antibodies, including COX-2, p38, extracellular regulated protein kinases (ERK)1/2, ICAM-1, c-Jun N-terminal kinase (JNK), phosphorylated-p38, phosphorylated-ERK1/2, phosphorylated-JNK (Cell Signaling Technology, MA, USA), NF-kappa-B inhibitor alpha (IκB-α), lamin B1, Nuclear factor erythroid 2-related factor 2 (Nrf2), HO-1, phosphorylated-IκB-α (Santa Cruz, CA, USA), and β-actin (Sigma). The membranes were incubated with secondary antibodies as appropriate. Finally, the membranes were treated with Luminol/Enhancer solution, and specific protein signals were detected using the BioSpectrum 600 system (UVP, Upland, CA, USA).
8
Protein Expression Analysis of Acacetin-Treated Cells
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Cells were treated with acacetin and lysed using protein lysis buffer (Sigma). Equal amounts of protein were separated on 8–10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA, United States). The PVDF membranes were blocked, incubated with primary antibodies overnight at 4°C, incubated with secondary antibodies, and the signal was detected with Luminol/Enhancer solution (Millipore). Protein bands were quantitated using the BioSpectrum 600 system (UVP, Upland, CA, United States). The primary antibodies included antibodies that recognized the following proteins: phosphorylated-AMPKα, AMPK, IκBα, phosphorylated-IκBα, p65, and lamin B1 (Santa Cruz, CA, United States); ERK1/2, phosphorylated-ERK1/2 (pERK1/2), JNK, phosphorylated-JNK (pJNK), p38, phosphorylated-p38 (pp38), fatty acid synthase (FAS), fatty acid binding protein (aP2), sirtuin 1 (Sirt1), SREBP-1c, and lipoprotein lipase (LPL) (Cell Signaling Technology, Danvers, MA, United States); acetyl CoA carboxylase-1 (ACC-1), phosphorylated-ACC-1 (pACC-1), adipose triglyceride lipase (ATGL), C/EBPα, C/EBPβ, PPAR-α, PPAR-γ, hormone-sensitive lipase (HSL), phosphorylated-HSL (pHSL) (Abcam, Cambridge, MA, United States); and β-actin (Sigma).
9
Regulation of NF-κB Signaling by A3AR
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RPMI1640 medium and fetal bovine serum (FBS) were purchased from GIBCO (MD, USA); human tumor necrosis factor-alpha (hTNF-α) was purchased from Cell Signaling Technology (MA, USA); antibodies to A3AR, NF-κB p65, IκB-α, and phosphorylated-IκB-α were purchased from Santa Cruz Biotechnology (CA, USA); secondary antibodies horseradish peroxidase-conjugated goat IgG were purchased from Beyotime (Jiangsu, China). 1-[2-Chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-β-D-ribofuranuronamide (2-Cl-IB-MECA) was purchased from Tocris Bioscience (Bristol, UK), and a stock solution of 100 mM was prepared in DMSO. The Cy 3-conjugated AffiniPure donkey anti-mouse IgG and fluorescein isothiocyanate- (FITC-) conjugated AffiniPure donkey anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (PA, USA); RNAiso Plus, PrimeScript RT MasterMix Perfect Real Time, and SYBR Premix Ex Taq II (Perfect Real Time) were purchased from Takara (Dalian, China). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Sangon Biotech (Shanghai, China).
10
Western Blot Antibody Panel
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β-actin, β-catenin, APC, Lamin B, COX-2, phosphorylated IκBα, NFκB p65, NFκB p50, Fas, Bax and survivin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cyclin D1, Cyclin E, CDK2, CDK4, Caspase-8, Bcl-2, p21, 15-PGDH and GSK3β antibodies were all purchased from Cell Signaling (Denver, MA).
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