Cells were lysed in RIPA buffer, and lysate was sonicated and then centrifuged for 10 min at 12,000 rpm to remove cell debris. Protein concentrations were determined using a BCA assay (Thermo Scientific, MA, USA). Equal amounts of proteins were then separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membrane was probed with an appropriate primary antibody and a secondary antibody conjugated to horseradish peroxidase. The following antibodies were utilized: β-actin (1:10,000 dilution, Abcam), Cleaved Caspase-3 (1:500 dilution; Cell signaling, MA, USA), 2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNPase, 1:1000 dilution, Chemicon), interleukin-1 receptor-associated kinase 1 (IRAK1, 1:1000 dilution; Santa Cruz), MBP (1:1000 dilution, Abcam), myelin proteolipid protein (mPLP, 1:1000 dilution, Abcam), NF-KB p65 (1:1000 dilution, Abcam), NG2 (1:1000 dilution, Santa Cruz), platelet-derived growth factor receptor α (PDGFR-α; 1:1000 dilution; Santa Cruz), TNF receptor associated factor (TRAF6, 1:1000 dilution; Santa Cruz). Proteins were visualized by enhanced chemiluminescence (Thermo Fisher Scientific).
Nf kb p65
Manufactured by Abcam
Sourced in United States
NF-κB p65 is a subunit of the NF-κB transcription factor complex. NF-κB plays a crucial role in regulating the immune response and is involved in cellular processes such as inflammation, immunity, cell growth, and apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (3 mentions)
Gapdh, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Irak1, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Traf6, by Santa Cruz Biotechnology (1 mentions)
Pdgfrα, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Proteinase and dephosphorylation inhibitors, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
P smad2, by Abcam (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
0.22 μm pvdf membrane, by Bio-Rad (1 mentions)
Tgf β, by Abcam (1 mentions)
T per reagent, by Thermo Fisher Scientific (1 mentions)
Multi beads shocker, by Thermo Fisher Scientific (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
A1r a1, by Nikon (1 mentions)
10 protocols using nf kb p65
1
Protein Extraction and Western Blot Analysis
2
Quantitative Western Blot Analysis of Lung Tissue
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Western blot was performed as previously described [25 (link)]. Briefly, lung tissue sample was homogenized using Multi-beads shocker® and added to the T-PER reagent (Thermo Fisher Scientific) consisting of proteinase and dephosphorylation inhibitors (Thermo Fisher Scientific). Total tissue protein purified from lungs were separated by SDS-PAGE gels and then transferred to 0.22-μm PVDF membranes (Bio-Rad). After blocking, the membranes were incubated with primary antibodies against NF-kB p65 (1:500 dilution, Abcam), IκBα (1:1000 dilution, CST), TGF-β (1:1000 dilution, Abcam), pSmad2 (1:1000 dilution, Abcam), α-Tubulin (1:1000 dilution, CST), or GAPDH (1:1000 dilution, Abcam), respectively; and followed by the appropriate horseradish peroxidase-conjugated secondary antibodies (Dako). The expression was visualized using an enhanced chemiluminescence detection kit (Thermo Scientific). Semiquantitative analysis was done using ImageQuant LAS 4000 mini (GE Healthcare Life Sciences).
3
Immunofluorescence and ROS Detection Assay
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Cells were washed with PBS 1X and fixed with PFA 4%. Immunofluorescence studies were performed by using antibodies against CD80 (eBioscience Inc.) for 1 h at 37 °C without permeabilization. Then, cells were washed with PBS 1X and slides were mounted and analysed with a confocal laser scanning microscope (Nikon A1R-A1). Image analysis was performed using the Nikon A1R-A1 software. Other immunofluorescence assays were performed by using antibodies against histone H2A.X (Genetex Inc.) and NF-kB p65 (Abcam) for overnight treatment at 4 °C after permeabilization. DRAQ5TM (Thermo Fisher) fluorescent probe solution was used to identify nuclei.
To test the presence of ROS, living cells were stained with 5 μM MitoSOX [3,8-phenanthridinediamine, 5-(6′-triphenylphosphoniumhexyl)-5,6 dihydro-6- phenyl] or 5 μM CM-H2DCFDA (all purchesad from Molecular Probes, Invitrogen, Carlsbad, CA) for 30 mins in CO2 incubator at 37 °C, after 30 mins and overnight treatment with pro-oxidants. For all these experiments, slides were mounted and analyzed with a confocal laser scanning microscope (Nikon A1R-A1 or Leica SP2). Image analysis was performed using the Nikon A1R-A1 software or Leica SP2 software.
To test the presence of ROS, living cells were stained with 5 μM MitoSOX [3,8-phenanthridinediamine, 5-(6′-triphenylphosphoniumhexyl)-5,6 dihydro-6- phenyl] or 5 μM CM-H2DCFDA (all purchesad from Molecular Probes, Invitrogen, Carlsbad, CA) for 30 mins in CO2 incubator at 37 °C, after 30 mins and overnight treatment with pro-oxidants. For all these experiments, slides were mounted and analyzed with a confocal laser scanning microscope (Nikon A1R-A1 or Leica SP2). Image analysis was performed using the Nikon A1R-A1 software or Leica SP2 software.
4
Protein Expression Analysis Protocol
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Protein was extracted using cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA). Protein content in the lysate was determined using the BCA Protein Assay (Pierce, Rockford, IL, USA). Equal amounts of protein were run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred to a polyvinylidene difluoride membrane. The following antibodies were used: matrix metalloproteinase (MMP)-1, SREBP-1, and Keratin 16 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), nuclear factor kappa B (NF-kB) p65, interleukin (IL)-1α, IL-8, IL-6, collagen1, adiponectin, and phosphorylated insulin- like growth factor 1 receptor (Abcam, Cambridge, MA, USA), phospho ERK, Akt, and PI3K (Cell Signaling Technology), MMP-7, and MMP-12 (Thermo, Pittsburgh, PA, USA). Secondary anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG antibody (Cell Signaling Technology) were used to detect primary antibodies. Films of blots were analyzed and quantified using a densitometric program (TINA, Raytest Isotopenmebgerate, Straubenhardt, Germany). All experiments were repeated a minimum of four times.
5
Investigating Ovarian Cancer Signaling
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Protein expressions of TRIM52, NF-kB P65, and NF-kB downstream effectors were tested by WB. Cell protein was extracted from stimulated ovarian cancer cells using RIPA buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, and 0.5% Na-deoxycholate) containing protease inhibitors. BCA protein assay kit (Thermo Fisher Scientific) was utilized to determine protein concentrations. Thirty micrograms samples of lysates were separated on 10–15% SDS-PAGE gels and transferred to PVDF membranes. The membranes were incubated with primary antibodies overnight at 4 °C. Primary antibody incubation was followed by incubation with an HRP-conjugated secondary antibody. Signals were detected using chemiluminescenct substrate (ECL, Bio-Rad, Richmond, CA, USA). Finally, the band intensity was measured using Image J software (NIH, Bethesda, MD, USA). Primary antibodies MMP9, Bcl2, caspase 3, IL8, TNFα, NF-kB P65, IKK, IKBα, and phosphorylated IKKβ and IKBα were purchased from Abcam, and antibodies against TRIM52 was from Novus. GAPDH and H3 serving as internal control were obtained from CST Biotech. (Danvers, MA, USA) and Abcam, respectively.
6
Antibody and Reagent Sources for Neuroinflammation
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Antibodies and reagents used in this work were purchased from the indicated sources: Lamp2 (NB300-591; Novus, Centennial, CO, United States); CD63 (ab216130; Abcam, Cambridge, MA, United States), TLR4 (NB100-56566 Novus, Centennial, CO, United States), Iba1 (Novus NB1001028 or Wako; 19-19741), STAT3 (4904S, Cell Signaling, MA, United States), ERK1/2 (9107S; Cell Signaling, MA, United States), goat anti-mouse-HRP (Santa Cruz Biotechnology; sc-2005), and goat anti-rabbit-HRP (Santa Cruz Biotechnology; sc-2004); NFkB p65 (16502, Abcam, Cambridge, MA, United States); MYD88 (ab2064; Abcam, Cambridge, MA, United States); β-Actin (A1978; Sigma-Aldrich, St. Louis, MO, United States). Cocaine hydrochloride (C5776) was purchased from Sigma-Aldrich, St. Louis, MO, United States. The RVG-Lamp2b plasmid was a gift from Drs. Seow Yiqi and Matthew Wood at the University of Oxford, Oxford, United Kingdom (Alvarez-Erviti et al., 2011 (link)).
7
Western Blot Analysis of NF-kB Pathway
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The extracted protein sample and appropriate loading buffer were heated at 95°C for 5 minutes. Then, 20 μg protein was used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, and 280 mA constant flow was transferred to membrane for 1.5 hours. 5% skimmed milk powder was used for blocking for 2 hours. After that, the primary antibody (NF-Kb-p65, Abcam, Cambridge, MA, USA, Rabbit, 1 : 2000, ab32536; Ikkα, Abcam, Cambridge, MA, USA, Mouse, 1 : 2000, ab178872; IkB-α, Abcam, Cambridge, MA, USA, Rabbit, ab32518; p- IkB-α, Abcam, Cambridge, MA, USA, Rabbit, ab133462; β-actin, Abcam, Cambridge, MA, USA, Mouse, 1 : 5000, ab8226) was incubated overnight at 4°C. The next day, after washing the membrane with tris-buffered saline-tween (TBST) 3 times, the horseradish peroxidase-conjugated secondary antibody (Abcam, Cambridge, MA, USA) was incubated for 2 hours at room temperature. After washing the membrane three times with TBST, the freshly prepared electrochemiluminescence (ECL) working solution (Gene, China) was added and exposed with a fully automatic imaging analyzer. Image J software was used to analyze the gray value of each band and finally performed the statistical analysis.
8
Immunohistochemical Staining and Quantification
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Immunohistochemical staining was performed using 2-mm paraffin-embedded tissue sections on Fisherbrand Superfrost Plus slides (Fisher Scientific, Waltham, MA, USA). Antibodies directed against 8-hydroxyguanosine (8-OHdG, 1:2000; Abcam, Cambridge, UK), malondialdehyde (MDA, 1:1000; Abcam), HMGB1 (1:100; Abcam), LC3A/B (1:50; Abcam), CD68 (1:50; Abcam), CD3 (1:100; Abcam), VCAM-1 (1:200; Abcam), and nuclear factor [(NF-kB p65 (1:2000; Abcam)] were used with the EnVi-sion+ HRP detection system (Dako, Glostrup, Denmark) with an ImmPRESSTM DAB Peroxidase Substrate kit (Burlingame, CA, USA). The percentage of positive staining was determined semiquantitatively using a scale ranging from 0 (-) to 4 (++++). Grading with approximate percentages indicating the number of relevant cells showing a positive reaction was as follows: 0) no specific immunohistologic reaction visible; 1) minimal (1-10% of relevant cells showing a weakly positive reaction; 2) mild (up to 25% showing a strong positive reaction); 3) moderate (up to 50% showing a strong positive reaction); 4) marked (more than 50% showing a strong positive reaction). Grades 2-4 were defined as positive, and 0 and 1 as negative. All photomicrographs were acquired using an Olympus Insight color camera (Tokyo, Japan).
9
Molecular Mechanisms of SIRT1 in Fibrosis
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Antibodies and other reagents SRT1720 was purchased from BioChemPartner (Shanghai, China). Antibodies against SIRT1, HIF1A, GLUT1, fibronectin, collagen IV, E-cadherin, α-SMA, NF-KB p65 and 8-hydroxydeoxyguanosine (8-OHdG) were obtained from Abcam. Antibodies for transforming growth factor beta 1 (TGFB1), NAD(P)H oxidase 4 (NOX4), CTGF, SNAIL, Histone H3 and β-actin were purchased from Proteintech (Chicago, USA). Goat anti-rabbit or mouse IgG-HRP secondary antibodies, TRITC-labelled goat anti-rabbit secondary antibody and FITC-labelled goat anti-rabbit or mouse IgG secondary antibodies were purchased from Zhongshan Goldenbridge Biotechnology (Beijing, China). Biochemical parameters in urine and plasma were measured with the reagent kits purchased from BioSino Biotechnology and Science (Beijing, China). PVDF membranes were purchased from Millipore. All culture media were from Gibco-BRL, and the Lipofectamine RNAiMAX was purchased from Invitrogen Life Technologies.
10
Kidney Protein Expression Analysis
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Kidney sample lysates were prepared with RIPA lysis buffer followed by centrifugation. Western blot analysis of IkBɑ (Santa Cruz, Dallas, TX, 1:500), Nrf2 (Santa Cruz, 1:500), HO-1 (Santa Cruz, 1:1000), NQO-1 (Santa Cruz, 1:1000), Bax (Santa Cruz, 1:1000), Bcl-2 (Santa Cruz, 1:1000), poly (ADP-ribose) (PAR) polymerase-1 (PARP-1) (Santa Cruz, 1:1000), b-actin (Abcam, Cambridge, UK, 1:10,000), Histone H3 (Santa Cruz, 1:500), p-NF-kB p65 (Cusabio, Wuhan, China, 1:500), p-IkBɑ (Cusabio, 1:500), NF-kB p65 (Abcam, Cambridge, UK, 1:1500), and GAPDH (Abcam, Cambridge, UK, 1:10,000) was performed according to standard protocols. The blots were detected by the Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, MA) followed by exposure to Kodak-X-Omat film (Shanghai, China).
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