BJAB B cells (5 × 106 per ml) with or without resveratrol treatment were lysed with RIPA buffer for standard western blotting using Abs specific to Sirt1, FcγRIIB (Santa Cruz), p65 NF-κB, acetylated-p65 NF-κB (K310), p65 NF-κB (S536), p65 NF-κB (S468) and β-actin (Cell Signaling Technology, Danvers, MA, USA). Immunoprecipitation was performed using 4 μg phosphor-p65 NF-κB (S536) or phosphor-p65 NF-κB (S468), which were separately incubated with 500 μg of pre-cleared whole cell lysates overnight before SDS-PAGE and western blotting. Four micrograms of each p65 NF-κB, phosphor-p65 NF-κB (S536), phosphor-p65 NF-κB (S468) and acetylated p65 NF-κB (K310) Abs (Cell Signaling) were used for chromatin immunoprecipitation. PCR was performed using sequence-specific primers to amplify the −600 to −300 promoter region of the Fcgr2b gene.
Nf κb p65
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China, Japan
NF-κB p65 is a protein subunit of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor complex. It plays a central role in regulating the immune response and inflammation.
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Lab products found in correlation
β actin, by Cell Signaling Technology (126 mentions)
P nf κb p65, by Cell Signaling Technology (109 mentions)
Phospho nf κb p65, by Cell Signaling Technology (81 mentions)
Gapdh, by Cell Signaling Technology (68 mentions)
P iκbα, by Cell Signaling Technology (61 mentions)
β actin, by Santa Cruz Biotechnology (44 mentions)
Erk1 2, by Cell Signaling Technology (43 mentions)
P jnk, by Cell Signaling Technology (37 mentions)
β actin, by Merck Group (37 mentions)
Cleaved caspase 3, by Cell Signaling Technology (37 mentions)
Bcl 2, by Cell Signaling Technology (36 mentions)
Pvdf membrane, by Merck Group (36 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (32 mentions)
Phospho nf κb p65 ser536, by Cell Signaling Technology (32 mentions)
Phospho iκbα, by Cell Signaling Technology (30 mentions)
P erk, by Cell Signaling Technology (29 mentions)
P akt, by Cell Signaling Technology (29 mentions)
Cox 2, by Cell Signaling Technology (29 mentions)
P38 mapk, by Cell Signaling Technology (28 mentions)
P erk1 2, by Cell Signaling Technology (26 mentions)
724 protocols using nf κb p65
1
Resveratrol Modulates NF-κB Signaling
2
Cell Signaling Pathway Analysis
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FBS, Horse Serum, antibiotic/antimycotic, EGF, insulin, Cholera Toxin, Hydrocortizone, L-Glutamine, HEPES, Sodium Pyruvate and media used in cell culture were purchased from Gibco, Invitrogen (Carlsbad, California). Etoposide and Doxorubicin were purchased from Tocris (Bristol, UK). The pan caspase inhibitor z.vad.fmk was purchased from Sigma (St. Louis, Missouri). TNFα, and PS-341 (Bortezomib) were purchased from Merck Millipore (Darmstadt, Germany). Protease inhibitor cocktail was obtained from Roche (Indianapolis, IN). Caspase −3, −7, −8, −9, PARP1, RIP1, Bid, TNFR1, TNFR2, AIF, FADD, p-IκBα, p-TRAF2, p65/NF-κB and α-Tubulin antibodies were purchased from Cell Signaling Technology (Danvers, Massachusetts). Total TRAF2, total IκBα, Histone H3, EGFR, and GAPDH antibodies were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). All other reagents were purchased from Sigma (St. Louis, Missouri).
3
Lipopolysaccharide-Induced Inflammation Signaling
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LPS from E. coli O111:B4 (LPS) and ammonium pyrrolidine
dithiocarbamate (APDC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Thiazolyl Blue tetrazolium bromide (MTT) was obtained from Alfa Aesar
(Haverhill, MA, USA). Antibodies against cyclooxygenase-2 (COX-2), phospho-p38
MAPK, p38 MAPK, phospho-extracellular signal-regulated kinase 1/2 (ERK1/2),
ERK1/2, phospho-c-Jun N-terminal kinase (JNK), JNK, c-Fos, c-Jun,
phospho-IκBα, IκBα, phospho-p65 NF-κB, p65
NF-κB, phospho-AMP-activated protein kinase (AMPK), AMPK,
phospho-acetyl-CoA carboxylase (ACC), ACC, and glyceraldehyde-3-phosphate
dehydrogenase were obtained from Cell Signaling Technology (Danvers, MA, USA).
The anti-inducible nitric oxide synthase (iNOS) antibody was obtained from
GeneTex (Irvine, CA, USA).
dithiocarbamate (APDC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Thiazolyl Blue tetrazolium bromide (MTT) was obtained from Alfa Aesar
(Haverhill, MA, USA). Antibodies against cyclooxygenase-2 (COX-2), phospho-p38
MAPK, p38 MAPK, phospho-extracellular signal-regulated kinase 1/2 (ERK1/2),
ERK1/2, phospho-c-Jun N-terminal kinase (JNK), JNK, c-Fos, c-Jun,
phospho-IκBα, IκBα, phospho-p65 NF-κB, p65
NF-κB, phospho-AMP-activated protein kinase (AMPK), AMPK,
phospho-acetyl-CoA carboxylase (ACC), ACC, and glyceraldehyde-3-phosphate
dehydrogenase were obtained from Cell Signaling Technology (Danvers, MA, USA).
The anti-inducible nitric oxide synthase (iNOS) antibody was obtained from
GeneTex (Irvine, CA, USA).
4
Quantifying Inflammatory Signaling Pathways
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Protein was extracted from cells with a buffer containing 1% SDS, 100 mM Tris-HCl, 1 mM PMSF, and 0.1 mM β-mercaptoethanol, following treatment with 0.5 μg/ml rat recombinant MIF (ProSpec) for 15 min, 30 min, and 60 min, respectively. Alternatively, protein was extracted from 1-cm spinal segments of the injured site at 0 day, 1 day, 4 days, and 1 week following contusion (n = 8 in each time point). Protein concentration of each specimen was detected by the Bradford method to maintain the same loads. Protein extracts were heat-denatured at 95 °C for 5 min, electrophoretically separated on 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were subjected to the reaction with a 1:1000 dilution of primary antibodies in TBS buffer at 4 °C overnight, followed by a reaction with the secondary antibody conjugated with goat anti-rabbit or goat anti-mouse HRP dilution 1:1000 (Santa Cruz) at room temperature for 2 h. After the membrane was washed, the HRP activity was detected using an ECL kit. The image was scanned with a GS800 Densitometer Scanner (Bio-Rad), and the data were analyzed using PDQuest 7.2.0 software (Bio-Rad). β-actin (1:5000) was used as an internal control. The antibodies used in Western blot are as follows: MIF (Abcam); p65NFκB (Cell Signaling Technology, CST), p-ERK1/2, and ERK1/2 (CST); and CD74 (Biorbyt) and β-actin (Proteintech).
5
Intestinal Inflammation Profiling in ApcMin Mice
6
Western Blot Analysis of MCPIP1 and NF-κB Signaling
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Treated cells were lysed using the Mammalian Cell Lysis kit (Sigma-Aldrich). Equal amounts of the proteins were electrophoresed in a sodium dodecyl sulphate-polyacrylamide gel (12%) under reducing conditions followed by transfer to polyvinylidene fluoride membranes. The blots were blocked with 5% non-fat dry milk in PBS. The western blots were then probed with antibodies recognizing the MCPIP1 (1:1,000, Santa Cruz), β-actin (1:1,000, Sigma-Aldrich) p65 NF-κB (1:200, Cell Signalling), β-catenin (1:200, Cell Signalling), Lamin B (1:1,000, Santa Cruz) The secondary antibodies were horseradish peroxidase-conjugated to goat anti mouse/rabbit IgG (1:5,000) as described previously43 (link). Full blots can be found in Supplementary Fig. 14 .
7
MTT Assay for NF-κB Activation
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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Nω-nitro-l -arginine methyl ester hydrochloride (L -NAME), polyethylene imidazole (PEI), and lipopolysaccharide (LPS: Escherichia coli 0111:B4) were purchased from Sigma Chemical Co. (St. Louis, MO). PP2 was obtained from Callbiochem (La Jolla, CA). The enzyme immunoassay (EIA) kit for determining PGE2 levels was purchased from R&D systems (Minneapolis, MN). A luciferase construct containing the binding promoter for NF-κB was purchased from Promega (Madison, WI). Fetal bovine serum (FBS), DMEM, and RPMI 1640 were obtained from Gibco (Grand Island, NY). RAW264.7 and human embryonic kidney (HEK) 293 T cells were purchased from the American Type Culture Collection (Rockville, MD). All other chemicals were of Sigma grade. Antibodies against total protein and phosphoprotein for Flag, p65/NF-κB, p50/NF-κB, Akt, IκBα, p85/PI3K, Syk, Src, lamin A/C, and β-actin were obtained from Cell Signaling Technology (Beverly, MA). Antibody against HA protein was purchased from Santa Cruz (Santa Cruz, CA).
8
Phytochemical Compound Procurement for Inflammation Study
9
Flow Cytometry and Immunofluorescence Analysis
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Flow cytometry analysis of cell lines in vitro was performed using anti-CD44-APC (Cat.#559942), anti-CD24-FITC (Cat.# 555427), and corresponding isotype antibodies (BD Biosciences) on a SY3200 (Sony Biotechnology) flow cytometer with a minimum of 10,000 live cells detected. Immunofluorescent staining of p65 NF-κB (Cell Signaling Technology) in BT474 PTEN− LTT cell line was performed in 4-well glass chambers slides (lab-tek) following pretreatment with SF for 2 hours and 30 min and 50 ng/ml TNF-α for 2 hours. Cells were fixed and permeabilized with methanol/acetone followed by blocking with 3% BSA. Nuclear staining was identified with 1 μg/ml DAPI and imaging was carried out with a Nikon Eclipse TE2000-S microscope and MetaMorph 7.6.0.0 (Molecular Devices).
10
Protein Expression Analysis via Western Blot
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Proteins were collected in a RIPA buffer (R0010, Solarbio, China) and separated by 12% SDS‒PAGE (Bio-Rad, China). After being transferred to PVDF membranes, blocked with 5% milk, the membranes were incubated with appropriate primary antibodies overnight at 4 °C. The primary antibodies included CTSK (1:800, #DF6614, Affinity, China), CD68 (1:1000, #14-0688-82, ThermoFisher), CD163 (1:1000, #14-1639-82, ThermoFisher), CD200R (1:1000, #DF4715, Affinity), p-p65 NF-κB (1:1000, #3039, Cell Signaling Technology), p65 NF-κB (1:1000, #3034, Cell Signaling Technology), and GAPDH rabbit antibody (1:1000, #5174, Cell Signaling Technology). After washing with TBS-0.01% Tween 20, the membranes were incubated with secondary antibody (1:1000, #14708, Cell Signaling Technology) for 2 h at 25 °C. After washing, the blots were visualized using an enhanced chemiluminescence reagent (D085075, Bio-Rad, China).
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