Western blotting analysis was performed as previously described [48 (link)]. The blot was probed with antibodies specific for iNOS (BD Biosciences), STAT1 (BD Biosciences), pSTAT1 (BD Biosciences), and β-actin (Sigma-Aldrich). Immunoprecipitation was done with anti-p65 and anti-p50 (Santa Cruz Biotech), as previously described [49 (link), 50 (link)]. The immunoprecipitated proteins were analyzed by Western blot analysis with anti-p65 (Santa Cruz Biotech).
Anti p65
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany, Canada, United Kingdom
Anti-p65 is a primary antibody that recognizes the p65 subunit of the NF-κB transcription factor. It is designed for use in various applications, such as Western blotting, immunoprecipitation, and immunohistochemistry, to detect and study the expression and localization of the p65 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti iκbα, by Santa Cruz Biotechnology (16 mentions)
Anti β actin, by Santa Cruz Biotechnology (14 mentions)
Anti gapdh, by Santa Cruz Biotechnology (14 mentions)
Anti cox 2, by Santa Cruz Biotechnology (11 mentions)
Anti β actin, by Merck Group (10 mentions)
Anti p50, by Santa Cruz Biotechnology (9 mentions)
Anti p p65, by Santa Cruz Biotechnology (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Anti p38, by Cell Signaling Technology (7 mentions)
Anti iκbα, by Cell Signaling Technology (7 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (7 mentions)
Anti inos, by Santa Cruz Biotechnology (6 mentions)
Anti lamin b, by Santa Cruz Biotechnology (6 mentions)
Anti phospho p65, by Cell Signaling Technology (6 mentions)
Anti jnk, by Santa Cruz Biotechnology (6 mentions)
Anti p38, by Santa Cruz Biotechnology (6 mentions)
Anti flag, by Merck Group (6 mentions)
Anti p iκb α, by Santa Cruz Biotechnology (5 mentions)
Anti phospho p65, by Santa Cruz Biotechnology (5 mentions)
Anti β actin, by Cell Signaling Technology (5 mentions)
150 protocols using anti p65
1
Western Blotting and Immunoprecipitation Analysis
2
Western Blot Analysis of Viral Proteins
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The purified empty or filled PVC particles were heated at 100°C for 10 min with 1× SDS loading buffer. The samples were then loaded on the Novex 4 to 20% tris-glycine protein gel for separation. The gels were transferred to nitrocellulose membrane (Millipore) using a Bio-Rad semidry blotter. Standard protocols for membrane probing were conducted to detect the protein bands of interest. The detections were performed by using goat anti-rabbit/mouse secondary antibody (horseradish peroxidase) and Pierce ECL Plus Western blotting substrate and then visualized by Tanon 5200 (Tanon). The following primary antibodies were used: anti-Flag (clone M2) monoclonal antibody (Sigma-Aldrich, F3165), anti–RNA polymerase β monoclonal antibody (Abcam, ab191598), anti-mRFP (Origene, TA180093), anti-p65 (C terminus, Santa Cruz Biotechnology, sc-372), anti-p65 (N terminus, Santa Cruz Biotechnology, sc-8008), anti–β-actin (Abcam, ab6276), anti-Akt (Cell signaling technology, 4685), and anti–Phospho-Akt Ser473 (Cell Signaling Technology, 4060). Anti-Pvc16, anti-Pnf, and anti-Pdp1 were generated from immunized rabbit serum using specific synthesized oligopeptides (Genscript).
3
Nuclear and Cytoplasmic Protein Extraction
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Total proteins from RAGE and/or CHI3L1 expression plasmid transfected cells were extracted using 0.1% NP-40 lysis buffer as previously described [35 (link)]. Nuclear proteins were extracted, incubating cells with hypotonic buffer (20 mM Tris-HCl pH 7,4, 10 mM NaCl, 3 mM MgCl2) for 15 minutes on ice and centrifuge at 3000 rpm for 10 min at 4°C. The pellet was then lysed with 1% NP-40 lysis buffer for 30 minutes on ice and supernatants were collected by centrifuging at 13,000 rpm for 30 minutes [35 (link)]. SDS-PAGE and Western blot were performed as previously described using anti-phospho/total STAT3 (Cell Signaling, Danvers, MA), anti-β-catenin (Abcam), anti-Histone H3 (Cell Signaling), and anti-p65 (Santa Cruz, Dallas, Tx) primary antibodies [36 (link)].
4
Evaluating Inflammatory Mediators
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Tissue culture reagents, such as Roswell Park Memorial Institute 1640 (RPMI1640) and fetal bovine serum, were purchased from Gibco BRL Co. (Grand Island, NY, USA). All chemicals were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Primary antibodies, including anti-iNOS, anti-COX-2, anti-pIκBα, anti-β-actin, anti-p65, and anti-PCNA, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); all the primary antibodies were rabbit source. Anti-rabbit secondary antibodies were purchased from Millipore (Billerica, MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-6 and TNF-α were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).
5
Investigating Inflammatory Signaling Pathways
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LPS (Escherichia coli, O127:B8) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mas receptor antagonist A779 was obtained from AbBiotech (San Diego, CA, USA). ACE2 inhibitor MLN-4760 was a product from EMD Millipore (Darmstadt, Germany). SB203580 (a specific inhibitor of p38 MAPK), PD98059 (a specific inhibitor of ERK1/2) and SP600125 (a specific inhibitor of JNK) were purchased from Santa Cruz Biotechnology (Delaware, CA, USA). Rabbit anti-ACE2, anti-p50, anti-p65, and mouse anti-phospho-p50, anti-phospho-p65 and anti-IκBα antibodies were procured from Santa Cruz Biotechnology. Rabbit anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-/JNK, anti-phospho-JNK, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, and horse anti-mouse IgG antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). TNF-α and IL-1β kits were purchased from Invitrogen (Eugene, OR, USA). AngII and Ang-(1-7) enzyme-linked immunosorbent assay (ELISA) kits were from Kamiya Biomedical (Seattle, WA, USA).
6
Modulation of Th17 Signaling Pathways
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After treatment for 2 h with or without etanercept (0.1 and 1 μg/mL) or adalimumab (1 and 10 μg/mL), cells were stimulated with the Th17-polarized panel and subsequently lysed with an equal volume of ice-cold lysis buffer (150 μl) 1 h later for MAPKs, p65-NFκB and STAT3 signalling and 24 h later for RORγt signalling. After centrifugation at 13,000 × g for 15 min, cell lysates were analysed by western blot with anti-MAPK (p38 and ERK), anti-phospho-MAPK (pp38 and pERK), anti-p65, anti-phospho-p65, anti-STAT3, anti-phospho-STAT3, and anti-RORγt antibodies (Santa Cruz Biotechnology, Santa Cruz, California, USA). The immunoreactive bands were visualized using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Sunnyvale, California, USA).
7
Immunofluorescence Assay for Transcription Factor p65
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The cells were fixed in 2% buffered paraformaldehyde at room temperature (RT) for 15 min, then permeabilized with methanol for 10 min, followed by two washes in phosphate-buffered saline (PBS). The samples were blocked by incubation for 60 min at RT with PBS containing 3% bovine serum albumin. The samples were incubated overnight at 4°C with anti-p65 (Santa Cruz, CA), followed by three 5-min washes with PBS. The bound primary antibody was detected by incubation for 45 min with the goat secondary antibody conjugated to Alexa 594 (dil. 1/1000, Molecular Probes) for 30 min at RT. The slides were washed three times in PBS and mounted under a coverslip using VectaShield mounting medium with DAPI (Vector Laboratories, Inc). All samples were visualized by confocal microscopy, which was performed on a Leica SP5 using a 40× dry objective lens. An emission/excitation laser for DAPI and Alexa 594 was used. A sample with no primary antibody was always included to control for background generated by the secondary Abs. The image processing was performed using PHOTOSHOP CS software (Adobe Systems, San Jose, CA).
8
Inflammatory Pathway Modulation in Cell Lines
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Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco BRL Co. (Grand Island, NY). All other chemicals, including lipopolysaccharide (LPS), cobalt protoporphyrin IX chloride (CoPP) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), were obtained from Sigma-Aldrich (St. Louis, MO). Primary antibodies such as anti-iNOS, anti-COX-2, anti-inhibitor kappa B (IкB)-α, anti-p-IкB-α, anti-p50, anti-p65, anti-actin and anti-proliferating cell nuclear antigen (PCNA) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Anti-p-extracellular signal-regulated kinase (ERK), anti-ERK, anti-p-c-Jun N-terminal kinase (JNK), anti-JNK, anti-p-p38, anti-p38, anti-p-protein kinase B (Akt) and anti-Akt were obtained from Cell Signaling Technology (Danvers, MA). Anti-HO-1 antibody was gained from Merck Millipore (Darmstadt, Germany), and anti-Nrf2 antibody was purchased from Abcam (Cambridge, MA). Anti-mouse, anti-goat and anti-rabbit secondary antibodies were supplied by Merck Millipore (Darmstadt, Germany). Tin protoporphyrin IX (SnPP IX), an inhibitor of HO activity, was obtained from Porphyrin Products (Logan, UT). The enzyme-linked immunosorbent assay (ELISA) kit for PGE2 was purchased from R&D Systems, Inc. (Minneapolis, MN).
9
LPS-induced oxidative stress analysis
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LPS serotype O111:B4 from E. coli was purchased from Sigma-Aldrich (St. Louis, USA, Cat. L2630). Antibodies were: anti-gp91phox from BD Pharmigen (Franklin Lakes, USA. Cat. 611414); anti-p65 from Santa Cruz Biotechnology (Dallas, Tex. Cat. sc-372); goat anti-mouse and donkey anti-rabbit secondary antibodies conjugated, respectively, with Alexa Fluor 555 or Alexa fluor 488 from Invitrogen (Carlsbad, USA. Cat. A21422, Cat.A21206); anti-CD31 from BD Pharmigen (San Diego, Calif. Cat. 550274); anti-TLR4 from Abcam (Cambridge, UK. Cat. ab22048). Dihydroethidium (DHE) was purchased from Molecular Probes (Eugene, Ore, Cat. D1168). The peptides gp91ds-tat [H]-R-K-K-R-R-Q-R-R-R-C-S-T-R-I-R-R-Q-L-NH2 and scrambled-tat [H]-R-K-K-R-R-Q-R-R-R-C-L-R-I-T-R-Q-S-R-NH2 were synthethized by Anaspec (San Jose, Calif) (24 (link)).
10
Subcellular Protein Extraction and Analysis
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Proteins were extracted using an NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific, Waltham, MA, USA) and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or western blotting analysis using standard procedures. Primary antibodies were as follows: anti-CBLB502 (Provided by Prof. Haifeng Song, Beijing Institute of Radiation Medicine), anti-p65, anti-Lamin A and anti-tubulin (Santa Cruz Biotechnology, Dallas, TX, USA).
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