HACs from six different donors were cultured in 24-well plates on glass coverslips (10,000 cells) and treated for 4 or 40 h with a medium supplemented with 200 U/ml IL-1α ± FBS-hBMSC-CM or XFS-hBMSC-CM at three different concentrations 1, 10, and 100 μg/ml, following an in vitro model well described by our group (Pereira et al., 2013 (link)). Cells were fixed in 3.7% PFA for 10 min at room temperature. After washing in D-PBS, cells were permeabilized with a solution containing 20 mM HEPES, 300 mM sucrose, 50 mM sodium chloride, 3 mM magnesium chloride, and 0.5% Triton X-100 for 10 min at 4°C. Non-specific binding was prevented by incubation with 20% goat serum (EuroClone) in PBS for 30 min at 4°C. Slides were incubated with a rabbit anti-NF-kB p65 antibody (1:400; Cell Signaling, Danvers, MA, United States) overnight at 4°C. After rinsing in D-PBS, a goat anti-rabbit fluorescence-labeled antibody Alexa fluor-488 (Life Technologies, Grand Island, NY, United States) was added for 30 min at 4°C. Cells were stained with 5 U/ml phalloidin-594 for 20 min to visualize the cytoskeleton. Coverslips were mounted with an aqueous mounting. Slides were observed at different magnifications and images acquired with the Axiovert 200M microscope. A quantification was also performed counting cell with positive nucleus on at least five different areas for each experiment.
Anti nf kb p65 antibody
Manufactured by Cell Signaling Technology
Sourced in United States, China
The Anti-NF-kB p65 antibody is a research-use only product that detects the p65 subunit of the NF-kB transcription factor. NF-kB p65 is a key regulator of gene expression involved in inflammatory and immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Anti atg5 antibody, by Cell Signaling Technology (2 mentions)
Anti flag hrp, by Cell Signaling Technology (2 mentions)
Clb buffer, by Cell Signaling Technology (2 mentions)
Anti mettl3 antibody 15073 1 ap, by Proteintech (2 mentions)
Anti ddit4 antibody, by Proteintech (2 mentions)
Anti ikbα antibody, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Goat serum, by Euroclone (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Gw9662, by Abcam (1 mentions)
Ox ldl, by Yuanye Bio-Technology (1 mentions)
Oil red o solution, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by ScienCell (1 mentions)
Goat serum, by Vector Laboratories (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti nf kb p65 antibody
1
NF-κB Translocation in Chondrocytes
2
Western Blot Analysis of Signaling Pathways
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Cells were washed twice with ice-cold PBS and ruptured with CLB buffer (Cell Signaling Technology) containing PMSF and cocktail inhibitor. Cell lysates were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) and then blotted. Specific antibodies used are listed below: anti-Mettl3 antibody (15073–1-AP, 1:500) was from Proteintech; anti-DDIT4 antibody (1:1000) was from Proteintech; antibody to IkBα phosphorylated at Ser32 (2859S, 1:1000), anti-IkBα antibody (9234S, 1:3000), antibody to P70S6K phosphorylated at T389 (9234S, 1:1000), antibody to AKT phosphorylated at T308 (4056S, 1:1000), antibody to p65 phosphorylated at Ser536 (3031S, 1:1000), anti-NFkB p65 antibody (6956S, 1:1000), anti-ATG5 antibody (12994S, 1:1000), anti-β-actin (3700S, 1:10000), anti-Flag-HRP (2044S, 1:20000) were from Cell Signaling Technology. All of the unprocessed scans of the blots were shown in the Source Data file.
3
Western Blot Analysis of Signaling Pathways
4
Curcumin Modulates Macrophage Inflammation
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The following materials were used in this study: PLLA-BVS crimped on 3.0 × 15 mm balloon catheters (VasoTech, Inc. Lowell, MA, USA), Tibetan miniature pigs purchased from Pearl River Laboratory (Dongguan, China), Anti-NF-kB p65 antibody (Cell signaling, #6956, USA.) and Anti-TNF alpha antibody for IHC (Abcom, #ab6671, UK.), (Poly(L-lactide) OH (Mw 15,000–30,000 Da, Daigang Biomaterial Company, #DG-LOH050, China), human acute monocytic leukemia cell line, THP-1 (ZhongqiaoXinzhou, China #ZQ0086), RPMI 1640 (Gibco, #11875085), FBS (Sciencell, #0500, USA), penicillin-streptomycin (Gibco, #15140122, USA), phorbol-12-myristae-13-acetate (PMA, MCE, Shanghai, China, #HY-1879), Oxidized LDL (Ox-LDL, Shanghai Yuanye Bio-Technology, #S24879-2mg, China), Oil Red O solution (Sigma,#SLBW7964, USA.), Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan).curcumin (MCE, Shanghai, China, #HY-N0005), GW9662 (Abcam, #ab141261, UK.)ELISA kit for IL-6, TNF-α, IL-10 and Lp-PL-A2 (Meimian Company, China), Trizol RNA extraction kit (Shanghai Shenggong Institute of Biological Engineering, China, #A019-2), PrimeScript™ RT kit with GDNA-Eraser reverse transcription Kit (TAKARA, #RR047A), SYBR qPCR Mix Kit (Guangzhou Sihe, China, #SH3005).
5
Quantifying NF-kB p65 Nuclear Translocation
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NF-kB p65 translocation into the nucleus, as an index of NF-kB activation, was measured using immunofluorescence staining. Cells were seeded onto chamber slides and grown into confluence. After exposures, slides were washed twice with ice-cold PBS, fixed with 3.7% paraformaldehyde, and permeabilized with 0.1% Triton X-100. Slides were blocked with 10% goat serum (Vector Laboratories, Burlingame, CA) for 1 h and incubated overnight at 4°C with anti-NF-kB p65 antibody (1:1000, Cell Signaling Technology Inc., Beverly, MA). The following day, slides were incubated at room temperature with anti-rabbit IgG antibody labeled with Alexa Fluor 555 (Molecular Probes, Eugene, Oregon, USA). Sections were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA) and visualized and photographed under a confocal laser scanning microscopy (Zeiss LSM510; Carl Zeiss, Thornwood, NY). The number of cells with p65 nuclear translocation were counted in six random fields in a masked fashion. Counts were expressed as a percentage of the number of translocated cells in comparison to that of total cells. Experiments were performed in triplicate and were repeated at least three times.
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