Lipopolysaccharide was purchased from Sigma-Aldrich (St Louis, Ca). Recombinant mouse IL-4, recombinant mouse IL-6, recombinant mouse IL-1β, recombinant mouse TNF-α, recombinant mouse IL-10, and recombinant mouse TGF-β were purchased from R&D Systems (Minneapolis, MN). Tocilizumab, LY2109761, R-7050, Stattic, Bay 11-7082, and SIS3HCl were purchased from Selleck (Huston, USA). IL-1 receptor antagonist (IL1Ra) was purchased from Make Research Easy (Nanjing, China). Monoclonal mouse anti-β-actin (TA-09) was purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Antibodies against OT, OTR, β-tubulin, CD206, F4-80, phospho-STAT3, STAT3, phospho-SMAD2, SMAD2, phospho-SMAD3, SMAD3, p65, and phospho-p65 were purchased from Abcam (Cambridge, UK) and Cell Signaling Technology (Danvers, MA). Secondary antibodies were purchased from Invitrogen Life Technology (Foster City, CA). Mouse ELISA kits were obtained from R&D Systems and CUSABIO (Wuhan, China). All reagents were analytical grade.
Phospho p65
Manufactured by Abcam
Sourced in United Kingdom, United States
Phospho-p65 is a reagent used in research applications. It is used for the detection and quantification of phosphorylated p65, a subunit of the NF-κB transcription factor complex. This reagent can be used in various experimental techniques such as Western blotting, ELISA, and immunohistochemistry to study the activation and regulation of the NF-κB signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Abcam (3 mentions)
β tubulin, by Abcam (2 mentions)
Phospho smad3, by Abcam (2 mentions)
Smad3, by Abcam (2 mentions)
Phospho p38, by Abcam (2 mentions)
Total p65, by Abcam (2 mentions)
Phospho iκbα, by Abcam (2 mentions)
β actin, by Abcam (2 mentions)
Tocilizumab, by Selleck Chemicals (1 mentions)
Smad2, by Abcam (1 mentions)
Recombinant mouse il 4, by R&D Systems (1 mentions)
Recombinant mouse il 6, by R&D Systems (1 mentions)
Recombinant mouse il 1β, by R&D Systems (1 mentions)
Recombinant mouse tnf α, by R&D Systems (1 mentions)
Recombinant mouse il 10, by R&D Systems (1 mentions)
Recombinant mouse tgf β, by R&D Systems (1 mentions)
Ly2109761, by Selleck Chemicals (1 mentions)
R 7050, by Selleck Chemicals (1 mentions)
Stattic, by Selleck Chemicals (1 mentions)
Sis3 hcl, by Selleck Chemicals (1 mentions)
11 protocols using phospho p65
1
Inflammatory Cytokine Regulation Analysis
2
Protein Expression Analysis by Western Blot
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Total cell and tissues lysates were prepared in 1× sodium dodecyl sulfate buffer. Identical quantities of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose filter membranes. The blots were incubated with antibodies specific for CBX6 (Abcam, CA, USA), S100A9 (Abcam, CA, USA), total-ERK1/2(Abcam, CA, USA), phospho-ERK1/2 (Abcam, CA, USA),total-p38(Abcam, CA, USA), phospho-p38(Abcam, CA, USA), total-p65 (Abcam, CA, USA), phospho-p65 (Abcam, CA, USA), total-p50 (Abcam, CA, USA), phospho-p50 (Abcam, CA, USA), or GAPDH (Abcam, CA, USA), after which they were incubated with IRDye 800-conjugated goat anti-rabbit IgG and IRDye 700-conjugated goat anti-mouse IgG, and the signals were detected using an Odyssey infrared scanner (Li-Cor). GAPDH was used as a loading control for these experiments.
3
Breast Cancer Cell Line Culture and Antibody Reagents
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Human breast cancer cells MDA-MB-231(cat# 300275) were obtained from Cell line service (CLS)-GmbH. MDA-MB-231-GFP was previously described19 (link). MDA-MB-231and MDA-MB-231-GFP were maintained in DMEM (Hyclone, Cramlington, UK). All media were complemented with 10% fetal bovine serum (FBS) (Hyclone, Cramlington, UK), 100 U/ml penicillin/streptomycin (Hyclone, Cramlington, UK). Human Umbilical Vein Endothelial Cells (HUVECs) (Cat # C-003-5C) were obtained from (Life Technologies, Invitrogen). HUVECs were maintained in MEM 199 supplemented with 20% FBS, penicillin/streptomycin, 2 mM L-glutamine, 5 U/ml heparin and 50 μg/ml endothelial cell growth supplements (BD Biosciences, Bedfrord, MA, USA). Human Lung Microvascular Endothelial Cells (HMVEC-L) (cat # CC-2527) were obtained from Lonza (Lonza, Walkersville, USA). HMVEC-L cells were maintained in EGM™-2-MV BulletKit (Lonza, Walkersville, USA). Antibodies to NF-κB, phospho-p65, TNF-α and iNOS were obtained from Abcam. Antibodies to STAT3, pSTAT3, β-actin, goat anti-mouse IgG-HRP (sc-2005) and goat anti-rabbit IgG-HRP (sc-2004) were obtained from Santa Cruz Biotechnology, Inc. LY294002 was purchased from Sigma-Aldrich.
4
Western Blot Analysis of Apoptosis Regulators
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For Western blotting analysis, the cells were harvested and lysed on ice for 30 min in RIPA buffer supplemented with protease inhibitors (100 mM Tris–HCl at pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X‐100, 1% deoxycholate acid, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 2 mM DTT, 2 mM leupeptin, 2 mM pepstatin). Cells lysates were centrifuged at 12,000 rpm for 15 min, and the supernatants were collected as total proteins. After the concentrations of protein samples were determined by the BCA method (Beyotime, Haimen, China), an equal amount of each sample was separated by SDS‐PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% nonfat dried milk solution for 2 h and incubated with primary antibodies, respectively. The antibodies used were against cl‐caspase 3, Bcl2, Kras, Hif1α, PHD2 (CST), phospho‐P65, total P65 (Abcam, Cambridge, MA) and β‐actin (Boster Bio Tec, Wuhan, China). After washing three times with PBST, the membranes were incubated with HRP conjugated secondary antibody and visualized with an ECL detection system. Protein expression was measured by ImageJ software.
5
Molecular Mechanisms of EMT Regulation
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Antibodies used in this study were anti-CBX1, E-Cadherin, N-Cadherin, Vimentin, Snail, pAKT, AKT, Twist, Slug, Zeb1, p65, phospho-p65, ERK, phospho-ERK, JNK, phospho-JNK, SMAD3, phospho-SMAD3, IGF-1R, IGF-1, EGFR, VEGFR1, FGFR1, KIT, PDGFR-α, TGFβ-R1, all sourced from Abcam; and MK-2206 (an AKT inhibitor), SC-79 (an AKT activator) from Selleck.
6
Western Blot Analysis of Innate Immune Signaling
7
Western Blot Analysis of Protein Expression
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Protein lysates were extracted from fresh tissue and cultures. They were separated on electrophoresis gels, followed by transfer onto polyvinylidene difluoride membrane (Millipore, Billerica, MA). Rabbit antibodies to keratin 17, EGFR, involucrin, phospho-p65 or β-actin (2 µg/ml; Abcam), rabbit antibodies to phospho-EGFR (Tyr845), phospho-IκBα or IκBα (2 µg/ml; Cell Signaling, Danvers, MA), and horseradish peroxidase-conjugated goat anti-rabbit IgG (1 µg/ml; Abcam) were used as the primary antibodies and secondary antibody, respectively. Signal was developed using an ECL chemiluminescence kit (Millipore). The band intensities were quantitated using ImageJ software and normalized to β-actin values accordingly.
8
Investigating NF-κB Signaling and Osteogenesis
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The RAW 264.7 cells were cultured with PBS, LPS, KR−1, and KR−2 media for 30 min, and MC3T3-E1 cells were grown in various CM osteogenic media for 14 days. Subsequently, the cells were lysed for 30 min in lysis buffer with protease inhibitors (MedChemExpress), and cell lysates were analyzed by gel electrophoresis after samples were collected for processing and transferred to nitrocellulose membranes. The membrane was incubated with the relevant antibody for 12 h at 4 °C before exposure, after blocking had been performed with a 5% bovine serum albumin solution (Solarbio) for 1 h. The following antibodies were used for Western blotting: p65, phospho-p65, IκBα, phospho-IκBα, GAPDH, β-tubulin, OCN, Runx-2, and COL1α1 (all 1:1000 dilutions, Abcam, Cambridge, UK).
9
Phospho-protein Analysis of Cell Signaling
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Cells were lysed in M-PER mammalian protein extraction reagent (Thermo Fisher, NY) containing protease inhibitor and tyrosine and serine-threonine phosphorylation inhibitors. Protein was separated on SDA-PAGE and transferred to nitrocellulose membrane. For phospho-protein analysis, extracted proteins were separated on Phos-tag gel (Wako) according to the manufacturer’s protocol. Immunoblotted proteins were detected using specific antibodies: phospho-p65 (Abcam, ab28856), total p65 (Cell Signaling, 4764), PP2A (Abcam, ab168350), GAPDH (Cell Signaling, 2118), Drebrin (Cell Signaling, 5202), tubulin beta-4 (Novus Biologicals, NBP1-57005), total tubulin beta (Cell Signaling, 86298), and beta-actin (Sigma, A5136). All primary antibodies were used at 1:1000 dilution.
10
Murine Macrophage Cell Line Protocols
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The American Type Culture Collection (ATCC; Rockville, MD, United States) provided the RAW264.7 murine macrophage cell line. Solarbio (Beijing, China) provided high glucose dulbecco’s modified eagle’s medium (DMEM). Gibco-BRL (Sydney, Australia) provided alpha modification of eagle medium (a-MEM) and fetal bovine serum (FBS). Dojindo (Kumamoto, Japan) provided cell counting kits (CCK-8). R and D (R and D Systems, Minneapolis, MN, United States) provided recombinant mouse RANKL and M-CSF. In addition, Sigma Aldrich (St Louis, MO, United States) provided the tartrate-resistant acid phosphatase (TRAP) staining kit and celastrol. Specific antibodies against extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, TAK1, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IkBα), p65, phospho-ERK (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), phospho-IkBα, phospho-p65, c-fos, NFATc1, and β-actin were obtained from Abcam (Cambridge, MA, United States).
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