The RPMI‐1640 or DMEM medium and fetal bovine serum (FBS) were purchased from Gibco, Inc. (Carlsbad, CA, USA). Phorbol 12‐myristate 13‐acetate (PMA) and lipopolysaccharide (LPS) from Escherichia coli (serotype 0111:B4) were obtained from Sigma‐Aldrich (St. Louis, MO, USA). Goat antimouse SDC1 antibody, and human tumour necrosis factor alpha (TNF‐α, Cat. DTA00C), interleukin‐1beta (IL‐1β, Cat. DLB50), cytokine‐induced neutrophil chemoattractant ELISA kits (CINC‐1, CXCL‐1, Cat. QC101) were all purchased from R&D Systems, Inc. (Minneapolis, MN, USA). NF‐κB P65 (8242) and NF‐κB p65 (3033) antibody were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Mouse anti‐human SDC1 antibody and CD15 antibody was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX ,USA) and ZSGB‐Bio Inc. (Beijing, China) respectively. Human Sdc1 ELISA kit was purchased from Elabscience Biotechnology Co., Ltd (Cat. E‐EL‐H1298; Wuhan, China).
Nf κb p65 8242
Manufactured by Cell Signaling Technology
Sourced in United States
The NF‐κB P65 (8242) is a rabbit monoclonal antibody that recognizes the p65 subunit of the NF‐κB transcription factor complex. This antibody is designed for use in applications such as Western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Lipopolysaccharide lps from escherichia coli serotype 0111 b4, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Bcl 2, by Wanlei (1 mentions)
β actin, by Wanlei (1 mentions)
Caspase 8 activity assay kit, by Beyotime (1 mentions)
Ac devd cho, by Beyotime (1 mentions)
Caspase 8, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Ao eb, by Beyotime (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Mccoy s 5a medium, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
5 protocols using nf κb p65 8242
1
Inflammatory Mediator Quantification in Cells
2
Protein Expression Analysis via Western Blot
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Western blot was used to detect the expression of protein. Membranes were blocked with 5% TBST diluted nonfat powdered milk at room temperature for 1 hour incubated with primary antibody (HMGB1, sc-56,698, santa cruz; TLR4S, sc-293,072 santa cruz; Phosphorylation of NF(P-NF)-κB p65, sc-166,748 santa cruz; Cleaved caspase3, #9961, Cell Signaling Technology; NF-κB p65 #8242, Cell Signaling Technology; bcl-2, WL01556, Wanlei; Bax WL01637 Wanlei; GAPDH WL01114; β-actin WL01372 Wanlei.) at 4 C for 14 h. β-actin and GAPDH were used as the standard proteins. The secondary antibody was diluted with blocking solution and incubated membranes for 90 mins at room temperature [29 (link)]. Image J was used to analyze the gray value of the protein band.
3
Apoptosis Induction in Cancer Cells
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McCoy's 5A medium, fetal bovine serum (FBS), penicillin-streptomycin and trypsin-EDTA were obtained from Gibco (Grand Island, NY, USA). NAC, Ac-DEVD-CHO, DAPI and AO&EB, DCFH-DA, JC-1 stain kit, Cell and Tissue mitochondria isolation kits, Caspase-8 activity assay kit and BCA protein assay kit were obtained from Beyotime Biotech (Jiangsu, China). Nuclear-Cytosol kit was obtained from Applygen Technologies Inc (Beijing, China). Annexin V-FITC/PI assay kit, ROS and TNF-α ELISA kits were purchased from KeyGEN Biotech (Nanjing, China). Antibodies against Bax (#2772), Bcl-2 (#4223), Cytochrome c (#11940), Caspase-3 (#9662), cleaved Caspase-3 (#9661), Caspase-8 (#4790), cleaved Caspase-8 (#9496), Caspase-9 (#9508), NF-κB (p65, #8242) and Histone H3 (#3638) were purchased from Cell Signalling Technology (Danvers, MA, USA). Anti-β-actin and anti-GAPDH antibodies, anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies, 5-Fluorouracil (5-FU), MTT and standard sugar were purchased from Sigma (St. Louis, MO, USA). All other chemicals used were of analytical grade.
4
Nuclear Protein Extraction and Immunoblotting
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Harvest and pellet BMDM cells. Resuspend cell pellet in 250 µL ice cold lysis buffer (10 mM Hepes pH 7.9 + 10 mM KCl + 0.1 mM EDTA + 0.4% Nonidet P40) with protease inhibitors (Roche, 1836145) and incubate on ice for 15 min. Spin the lysate for 1 min at full speed at 4°C. Transfer supernatant to a fresh tube to keep the cytoplasmic fraction. Wash the nuclear pellet in 500 µL ice cold lysis buffer and spin down for 1 min at 4°C. Add 20 µL extraction buffer (20 mM Hepes pH 7.9 + 0.4 M NaCl + 1 mM EDTA) with inhibitors to the pellet and shake vigorously for 15 min on vortex in cold room. Spin for 10 min at full speed at 4°C. Transfer supernatant to fresh tube and store this nuclear extract at −20°C. Denature protein before use. Immunoblotting was performed as described previously (27 (link)). Anti-IκBα (9247), -phospho-IκBα (9246), -ERK (4372), -phospho-ERK (4695), and -NF-κB-p65 (8242) antibodies were obtained from Cell Signaling (Danvers, MA, USA). Anti-β-actin (ab8224) and -PCNA (ab152112) antibodies were from Abcam (Cambridge, MA, USA).
5
Preparation and Analysis of Cell Lysates
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Total cell lysates were prepared in a lysis buffer containing 50 mM tris-HCl (pH 7.8), 137 mM NaCl, 10 mM NaF, 1 mM EDTA, 1% Triton X-100, 10% glycerol, and the protease inhibitor cocktail (Roche) after three freeze/thaw cycles. Tissue lysates were prepared by homogenizing in a buffer containing 50 mM tris (pH 7.6), 130 mM NaCl, 5 mM NaF, 25 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and the protease inhibitor cocktail. The antibodies used are as follows: rabbit anti-IκBα (4812), rabbit anti-β-TrCP (4394), phospho–NF-κB–p65 (S536 and 3033), and NF–κB–p65 (8242), which were purchased from Cell Signaling Technology. Mouse anti-Flag (F3165), mouse anti-myc (M5546), and mouse anti-tubulin (T6199) were purchased from Sigma-Aldrich. Rabbit anti–hemagglutinin (sc-805) was purchased from Santa Cruz Biotechnology.
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