Human dermal fibroblasts were grown overnight in complete medium as previously described (You et al., 2010 (link)). Cells were trypsinized and placed in serum-free medium prior to plating onto different fibronectin substrates (pFn alone or pFn mixed with EDA) (Shinde et al., 2015 (link)). Cells appeared equally adherent and well spread on both substrates. Incubation times and doses of inhibitors are listed in the respective figure legends. The TLR4 (TAK242) and NFκB (BAY11–7082) inhibitors were purchased from EMD Millipore (Billerica, MA). Blocking antibody to α4 (MAB16983) was from Millipore (Temecula, CA). Blocking antibody to human TLR4 was obtained from R&D Systems (Minneapolis, MN). Subsequently, total RNA was isolated from fibroblasts using RNeasy extraction kit (Qiagen, Valencia, CA). An RT2 First Strand kit (Qiagen) was used to convert 1.5 μg of RNA into cDNA. The cDNA was applied to either an Extracellular Matrix and Cell Adhesion Molecule Array or a Fibrosis Array (Qiagen). A MyiQ cycler system (Bio-Rad Laboratories, Hercules, CA) was utilized for real-time PCR detection. Gene expression profiling was analyzed using an Excel-based PCR array data analysis template provided by the manufacturer. Relative gene expression was calculated as the difference in fold-change upon treatment and normalized against 5 housekeeping genes using the ΔΔCt method.
Nfκb bay11 7082
Manufactured by Merck Group
Sourced in United States
NFκB (BAY11–7082) is a lab equipment product. It is a potent and specific inhibitor of the transcription factor NF-κB. NFκB plays a critical role in the regulation of various cellular processes, including immune response, inflammation, and cell survival.
Automatically generated - may contain errors
Lab products found in correlation
Myiq cycler system, by Bio-Rad (1 mentions)
Mab16983, by Merck Group (1 mentions)
Rt2 first strand kit, by Qiagen (1 mentions)
Rneasy extraction kit, by Qiagen (1 mentions)
Sp600125, by Merck Group (1 mentions)
Sb203580, by Merck Group (1 mentions)
Pd98059, by Merck Group (1 mentions)
Anti tlr2 nb100 56726, by Novus Biologicals (1 mentions)
Mab003, by R&D Systems (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Endotoxin removal kit, by GenScript (1 mentions)
4 protocols using nfκb bay11 7082
1
Fibroblast Transcriptome Analysis on Fibronectin Substrates
2
Immunoblotting and Immunofluorescence Assays
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Unless indicated otherwise, all reagents were purchased from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). Monoclonal mouse antibodies against phospho-p38 MAP- kinase, NFκB and GAPDH were from Cell Signaling Technology (Danvers, MA). Antibodies to phospho- JNK (Thr183/185), and lamin A/C were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody to NF-κB phospho (S536) p65 used for tissue array staining was from Abcam (Cambridge, MA). Alexafluor488-conjugated secondary anti-mouse IgG (H+L) antibody was from Molecular Probes (Eugene, OR). Horseradish peroxidase (HRP) conjugated secondary antibodies to mouse IgG (H+L) and rabbit IgG (H+L) were from BioRad (Berkeley, CA). Antibodies against the 40kDa (gelatin-binding) region of fibronectin were described previously 13 (link). The functional upstream domain (FUD) of streptococcus pyogenes adhesin F1 protein was described previously 14 (link). The TLR4 (TAK-242) and NF-κB (BAY11-7082) inhibitors were from EMD Millipore (Temecula, CA). Recombinant His-tagged fibronectin Type III domains were described previously 15 (link).
3
Chondrocyte-Osteoclast Co-culture Protocol
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The pretreatments of chondrocytes before starvation were the same as described above. After a 16-h starvation, the culture medium was replaced with fresh 1% FBS DMEM in the mono-culture group or into the 1:1 (V/V) mixed media (osteoclast-conditioned media and 1% FBS DMEM) in the co-cultured group. The specific inhibitors of MAPKs (ERK: PD98059 50 μmol·L−1, p38: SB203580 20 μmol·L−1 and JNK: SP600125 20 μmol·L−1, purchased from Sigma, St. Louis, MO, USA) and nuclear factor-κB (NF-κB; Bay11-7082, 20 μmol·L−1, from Sigma, St. Louis, MO, USA) were then immediately added into the mono-culture and co-culture groups, respectively. Media samples (300 μL) were collected at 12, 24, 48 and 72 h for zymography. Samples of 1 000 μL cell lysate were collected at 2 h for semi-quantitative PCR. To study the role of IL-1β in inhibitor-pretreated chondrocytes, IL-1β was added into the mono-culture and co-culture chondrocytes after 30 min of incubation with inhibitor.
4
Molecular Expression and Mutagenesis of Mycobacterial HBHA
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The pET30a vector (in our laboratory) was used as an expression vector for N. cyriacigeorgica GUH-2 HBHA in E. coli. The plasmid pK18mobsacB (in our laboratory) was used to construct the hbha deletion mutant. Anti-TLR4 (NB100-56727, Novus Biological, USA), anti-TLR2 (NB100-56726, Novus Biological, USA), and IgG2A isotype control (MAB003, R&D, USA) were used in the present study. The following antibodies were purchased from Cell Signaling Technology (Danvers, USA): anti-p-Jnk (4668), anti-p-ERK1/2 (4370), anti-p-p38 (4511), anti-p-p65 (3033), and anti-β-actin (7074). The following pharmacological inhibitors were purchased from Sigma-Aldrich (St. Louis, MO, USA): p38 (SB203580), ERK1/2 (PD98059), JNK (SP600125), and NF-κB (BAY 11-7082). The endotoxin removal kit was purchased from GenScript (Nanjing, China). Human and mouse TNF-α, IL-6, and IL-10 ELISA kits were used in this study (BD, USA). DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride) and Alexa Fluor® 488 donkey anti-mouse IgG were purchased from ThermoFisher Scientific (Carlsbad, CA, USA). The adjuvant was purchased from Biodrago (Suzhou, China).
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