Cells covered with PBS were exposed to 40 mJ/cm2 UVB (emission peak at 302 nm) using a CL-1000 M UV crosslinker (UVP, Upland, CA, USA), and the intracellular oxidative stress of Hs68 cells was quantified using DCFDA fluorogenic dye (Sigma-Aldrich, Saint Louis, MO, USA). Fluorescence was detected using a microplate reader (Synergy HTX, BioTek Instruments, Winooski, VT, USA) at emission and excitation wavelengths of 520 and 488 nm, respectively. For immunofluorescence staining, HS68 cells were fixed on coverslips with 4% paraformaldehyde after exposure to 40 mJ/cm2 UVB. The slips were blocked with 5% skim milk in Tris-buffered saline (pH 7.6) containing 0.3% Triton X-100 and incubated with the following primary antibodies: anti-NFκB antibody (Cell Signaling, Danvers, MA, USA) and anti-4, 6-diamidino-2-phenylindole (DAPI) antibody (Cell Signaling, Danvers, MA, USA) for 30 min at 20 ± 1 °C. Following washing with PBS, the slips were incubated with the secondary-antibody anti-rabbit immunoglobulin (IgG) (Alexa Fluor 488, Invitrogen, Carlsbad, CA, USA) for 30 min at 20 ± 1 °C. The cover slips were counterstained with ProLong Gold anti-fade reagent and DAPI (Thermo Fisher Scientific, Waltham, MA, USA), and images were captured using a fluorescence microscope (Leica DMIL, Wetzlar, Germany).
Anti nf κb antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-NF-κB antibody is a laboratory reagent used to detect and study the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) protein. NF-κB is a transcription factor that plays a key role in regulating the immune response, inflammation, and other cellular processes. This antibody can be used in various experimental techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to analyze the expression and localization of NF-κB in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho nf κb antibody, by Cell Signaling Technology (3 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
Cl 1000m uv crosslinker, by Analytik Jena (1 mentions)
Anti rabbit immunoglobulin igg alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Synergy htx, by Agilent Technologies (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Protran ba 83, by GE Healthcare (1 mentions)
Anti rabbit igg conjugated with alexa 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Vector Laboratories (1 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Recombinant tgf β1, by R&D Systems (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody, by Beyotime (1 mentions)
Anti hmgb1 antibody, by Abcam (1 mentions)
Anti aqp4 antibody, by Abcam (1 mentions)
Anti histone h3 antibody, by Beyotime (1 mentions)
Fps zm1, by Merck Group (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Axiocam hrm camera, by Zeiss (1 mentions)
Axioimager microscope, by Zeiss (1 mentions)
Antipain, by Merck Group (1 mentions)
17 protocols using anti nf κb antibody
1
UVB-induced Oxidative Stress and NF-κB Activation
2
NF-κB Signaling in RA T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD4+ T cells were prepared from PBMC of 3 healthy donors (male to female 1:2) and 3 patients with RA (male to female 1:2). We also used CD4+ T cell line Jurkat as a positive control. Immunoblotting was performed as described previously [13 (link)]. Briefly, whole-cell extracts were prepared from lysis of PBMC in TNE buffer (50 mM Tris-hydrochloride [pH 7.4], 0.5% [v/v] Nonidet P-40, 150 mM sodium chloride, 5 mM ethylenediaminetetraacetic acid, 50 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, and 10 mg/mL aprotinin). Proteins (7 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Protran BA83; GE Healthcare, Chalfont St. Giles, UK) for blocking. The membranes were incubated with anti-phospho-NF-κB antibody (Ser 536, #3033; Cell Signaling Technologies, Danvers, MA), anti-NF-κB antibody (#8242; Cell Signaling Technologies), anti-UBASH3A antibody (GTX116432; GeneTex; Irvine, CA), and anti-β actin antibody (A1978; Sigma-Aldrich, St. Louis, MO). The bound antibodies were visualized with secondary antibodies against mouse and rabbit immunoglobulin G (IgG, GE Healthcare) conjugated with horseradish peroxidase and chemiluminescence reagent (ECL™ Prime western blotting Detection Reagent; GE Healthcare).
3
NF-κB Immunostaining in HUVECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HUVECs were grown on 0.2% gelatin coated small coverslips. Cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) after the completion of the experiment. 4% paraformaldehyde (PFA) was used for fixation of cells followed by 2 times washing with DPBS for 10 min each. Next, 50 mM of ammonium chloride (NH4Cl) in DPBS was used for blocking for 10 min at room temperature (RT). Cell permeabilization was done with 0.2% Triton X-100 (Sigma, St. Louis, MO, USA) in DPBS buffer (3 times 4 min each), followed by incubation with anti-NF-κB antibody (Cell Signaling, Leiden, The Netherlands. cat no, mAb #8242), 1:100 dilution in permeabilization buffer at RT for 1 h. Afterward, cells were again washed 3 times for 4 min each with 0.2% Triton X-100 in DPBS. Cells were incubated with a secondary antibody anti-rabbit IgG conjugated with Alexa 488 (Life Technologies, Darmstadt, Germany), at 1:300 for 1 h at RT. Excessive antibody was removed by washing again with 0.2% Triton X-100 in DPBS (3 times 4 min). Finally, cells were mounted with 4′,6-diamidino-2phenylindole, dihydrochloride (DAPI) (Vector Laboratories, Newark, NJ, USA). Axiovert 200M confocal Microscopy (Carl Zeiss, Jena, Germany) was used to visualize the fluorescent cells.
4
TGF-β1 Induces NF-κB Binding Profile
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 cells was stimulated by 5 ng/ml of recombinant TGF-β1 (R&D Systems). After 24 h, cells were used to perform RNA immunoprecipitation (RIP) experiments using an anti-NF-κB antibody (Cell Signaling Technology, CST) and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. The RNA fraction isolated by RIP was analyzed by qRT-PCR.
5
NF-κB Transcription Factor ChIP Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human MDA-MB 231 and BT-549 TNBC cells were seeded in 15-cm culture dishes and treated with TQ (50 μM) for 0, 2, 4, 6, and 8 h. At the end of the treatment period the cells were fixed with 1% formaldehyde at room temperature for 10 min and neutralized with glycine. The cells were collected, resuspended in CHIP lysis buffer, and sonicated (Vibra-Cell,TM Newtown, CT, United States). Samples were incubated with protein-G beads that had been pre-incubated with 4–10 μg of anti-NF-κB antibody (Cell Signaling Technology, MA, United States) or negative control IgG (Sigma-Aldrich Co., St. Louis, MO, United States). The next day, immunoprecipitants were washed by using washing buffer and reverse cross-linked at 65°C. The DNA was then purified by using a PCR purification kit purchased from MACHEREY-NAGEL (Duren, Germany). Finally, the purified DNA was subjected to quantitative RT-PCR and data analyzed.
6
Tanshinone IIA Modulates HMGB1-Mediated Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Special reagents and antibodies included recombinant HMGB1 (rHMGB1, ProSpec, Rehovot, Israel), IL-6 (PeproTech, NJ, USA), FPS-ZM1 (Sigma–Aldrich, MO, USA), anti-HMGB1 antibody (Abcam, Cambridge, UK), anti-AQP4 antibody (Abcam), anti-NF-κB antibody (Cell Signaling Technology, MA, USA), anti-IL-6 antibody (Millipore, MA, USA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Beyotime, Shanghai, China), anti-histone H3 antibody (Beyotime), and anti-Cadherin antibody (Beyotime). Tanshinone IIA was sourced from IMAM International Pharmaceuticals, Ltd. (Tianjin, China).
7
Immunohistochemical Analysis of NF-κB in Lung Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunohistochemistry analysis, the paraffin-embedded sections of lung tissues were deparaffinized with xylene and then rehydrated. Section slides were incubated with 3% hydrogen peroxide for 10 min, then in 5% BSA in PBS blocking solution for 20 min, and after, incubated overnight with anti-NF-κB antibody (Cell signaling Technology) in blocking solution at 4 °C. After washing with PBS, the slides were treated with biotinylated secondary antibody for 20 min, streptavidin-HRP (horseradish peroxidase) for 20 min, and 3,3N-Diaminobenzidine Tetrahydrochloride for 10 min. The slides were then washed, and counter stained with hematoxylin. Slides were evaluated by microscopy, and the positive cells exhibited yellow or brown particles.
8
Immunofluorescence Imaging of NF-κB
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The procedure was performed as previously described (30 (link)). Raji cells or Jurkat cells were fixed with 3% formaldehyde in PBS for 20 min at room temperature. After three washes with PBS containing 50 mM NH4Cl, the cells were soaked in a blocking solution (PBS containing 5% fetal calf serum, FCS; Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China) for 15 min at room temperature and then permeabilized with 0.1% Triton X-100 in PBS for 5 min. Cells were incubated with anti-NF-κB antibody (1:1,000; cat no. 8242; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C, and stained with a rhodamine-fluorescence labeled goat anti-rabbit secondary antibody (1:1,000; cat no. 15076; Active motif, Inc., Carlsbad, CA, USA) in 1% blocking solution (PBS containing 1% FCS) and incubated for 1 h at room temperature. Confocal imaging was performed with an LSM META510 confocal scanning laser microscope (Carl Zeiss AG, Oberkochen, Germany) at ×1,000 magnification. The images were subsequently analyzed using the freely available image processing software ImageJ Version 1.46 (National Institute of Health, Bethesda, Maryland, USA). The observations were made in triplicates, and representative images are presented here.
9
Colonoid Monolayer NF-κB Immunostaining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For colonoid monolayer immunostaining, after 30 min of stimulation, coverslips were fixed in 4% PFA for 15 min at room temperature. Then the cells were rinsed in PBS 1X twice, and treated with PBS, 0.1% Triton X-100 and 0.05% Tween 20 for 15 min, then blocked with 2% donkey serum in PBS, 0.01% Triton X-100 and 0.05% Tween 20 overnight. Coverslips were then stained with anti-NFκB antibody (1:100; Cell Signaling #8242) overnight at 4 °C followed by secondary antibody staining with anti-rabbit Alex Fluor-4588 and Alexa Fluor 468-phalloidin for 60 min, washed and mounted using ProLong Gold Antifade reagent containing 4′,6-diamidino-2-phenylindole (DAPI) for DNA staining. Sections were viewed on a Zeiss AxioImager microscope and images taken using an AxioCam HRm camera operating through Zen software.
10
Profiling Phosphorylated Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were extracted with a lysis buffer (20 mM Tris-HCl (pH = 8.0; FUJIFILM Wako), 150 mM NaCl (FUJIFILM Wako), 2 mM EDTA (FUJIFILM Wako), 100 mM NaF (FUJIFILM Wako), 1% NP40 (FUJIFILM Wako), 1 μg/mL leupeptin (Sigma), 1 μg/mL antipain (Sigma), and 1 mM phenylmethylsulfonyl fluoride (Sigma)). Phosphorylated protein and total protein in these lysates were examined by western blotting assay by using following primary antibodies: anti-phospho-Met (Tyr1234/1235) antibody, anti-phospho-Met antibody (Tyr1349), anti-Met antibody, anti-phospho-ERK1/2 antibody, anti-ERK1/2 antibody, anti-phospho-Akt antibody, anti-Akt antibody, anti-phospho-p38MAPK antibody, anti-p38MAPK antibody, anti-phospho-NF-κB antibody, anti-NF-κB antibody (Cell Signaling Technology, Beverly, MA, USA), anti-RANKL antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin antibody (Sigma). Proteins were visualized using Luminata Forte (Merck Millipore, Nottingham, UK) according to the manufacturer’s instructions.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!