Kidney samples were homogenized with RIPA lysis buffer and protein was extracted from the sample by centrifuging at 10 000 rpm for 15 min at 4°C. BCA method was used for the estimation of total protein in the sample as per the instructions in the kit. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate protein from the supernatant solution equivalent to 50 μg of protein. Protein was poured on the membrane and then tris buffer was used to block binding to the membrane for 60 min. Rabbit polyclonal anti-NF-κB p65 (1: 1000), anti-inducible NO synthase (iNOS) [1: 1000], anti-Akt (1: 1000), and anti-pAkt (1: 1000) antibody (Proteintech Group, Inc, Wuhan, China) were incubated with the blots at 4°C for overnight. Later, secondary rabbit anti-mouse IgG horseradish peroxidase (HRP) antibody (Proteintech Group Inc, Wuhan, China) was used to incubate for 60 min at room temperature after washing the membrane with TBST. An ECL detection system was used for development and detection of blots.
Anti nf κb p65
Manufactured by Proteintech
Sourced in United States, China
The Anti-NF-κB p65 is a research-use-only antibody that specifically recognizes the p65 subunit of the NF-κB transcription factor complex. NF-κB is a key regulator of immune and inflammatory responses in cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Anti lamin b1, by Proteintech (2 mentions)
Anti iκbα, by Proteintech (2 mentions)
Anti traf1, by Proteintech (2 mentions)
Anti β actin, by Proteintech (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Beyotime (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Anti akt, by Proteintech (1 mentions)
Penicillin streptomycin mix, by Thermo Fisher Scientific (1 mentions)
Tris buffered saline (tbs), by Thermo Fisher Scientific (1 mentions)
Anti nrf2 antibody, by Proteintech (1 mentions)
Ifn γ, by Proteintech (1 mentions)
Tnf α, by Proteintech (1 mentions)
Pyrrolidine dithiocarbamate, by Merck Group (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Anti cd31 antibody, by BD (1 mentions)
Cytodex 3 beads, by Cytiva (1 mentions)
P iκbα, by Proteintech (1 mentions)
13 protocols using anti nf κb p65
1
Kidney Protein Extraction and Western Blot
2
Evaluating Date Seed Bioactivity
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PVDF layers (Immobilon-P, 0.45 μm), dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were acquired from Sigma-Aldrich (Saint Louis, MO, USA). The following items were acquired from Thermo Fisher Scientific: Penicillin-streptomycin mix, Dulbecco’s altered Eagle’s medium (DMEM), trypsin (0.25% EDTA), fetal bovine serum (FBS), tris-buffered saline (TBS), and phosphate-buffered saline (PBS) (Shanghai, China). Proteintech Group, Inc. sold anti-NF-κB p65, anti-lamin B1, and anti-Nrf2 antibodies for rabbits. Bioscience Technology provided sodium lauryl sulphate, tris base, acrylamide, glycine, ammonium persulfate, bromophenol blue, and N,N’-methylene acrylamide (Shanghai, China). Proteintech provided TNF-α, IFN-γ, IL-6, and IL-1β enzyme-allied immunosorbent test (ELISA) implements (Wuhan, China). Date seeds of the Siwa variety were acquired from the Shalli Company (Siwa, Marsa Matruh, Egypt) in March 2022; the seeds were harvested at room temperature 12 h prior to arriving at our lab where they were then prepared immediately without being stored. All the other substances employed in this investigation, including the solvents, were of analytical grade.
3
Angiogenesis Inhibition Assay Protocol
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Ethanol, fibrinogen, aprotinin, thrombin and Pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma Chemical Co. (St. Louis, MO). Cyanidin-3-glucoside (C3G) was prepared as previously described [38 (link)]. Anti-MCP1 and anti-VEGF antibodies were obtained from Abcam (Cambridge, MA). Anti-NF-κB p65, IκBα, p-IκBα and anti-LMNB1 antibodies were purchased from Protein Tech Group (Chicago, IL, USA). Anti-β-actin was obtained from Cell signaling Technology (Danvers, MA). Anti-CD31 antibody was obtained from BD Pharmingen (San Diego, CA). Reactive oxygen species detection reagents were obtained from Invitrogen Molecular Probes (Eugene, OR). MTT assay kit was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Matrigel Invasion Chambers were purchased from BD Biosciences (Bedford, MA). Cytodex 3 beads were purchased from Amersham Pharmacia Biotech (Piscataway, NJ).
4
Protein Quantification and Western Blot Analysis
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Total proteins were extracted from the infarct cortex and protein concentrations were determined using a BCA assay kit as instructed by the manufacturer (Beyotime Biotech, Jiangsu, China). 1/4 volume of 5x loading buffer was added and followed by a boiling water bath for 10 minutes for denaturation. Samples, containing 40 µg proteins, were loaded and separated by electrophoresis on 12% SDS-polyacrylamide gels. Subsequently, proteins were transferred onto PVDF membranes, blocked for two hours with 5% nonfat dry milk at room temperature, and incubated with rabbit anti-TLR4 (1 : 300, Abcam, Cambridge, MA, USA), anti-NF-κB p65 (1 : 2000, Proteintech, Rosemont, USA), anti-IκBα (1 : 2000, Proteintech, Rosemont, USA), anti-β-actin (1 : 200, Boster, Hubei, China), and anti-Histone 3 (1 : 2000, Proteintech, Rosemont, USA) antibodies at 4°C on a shaker overnight and then washed, followed by incubation with the corresponding HRP-conjugated secondary antibody (Boster, Hubei, China). Finally, ECL solution was added to reveal the bands and the gray value was analyzed by ImageJ software (NIH).
5
NF-κB p65 Immunofluorescence Staining
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Cells were seeded on glass slides, fixed with 4% paraformaldehyde (Sinopharm Chemical Reagent, Beijing, China) for 15 min, and permeabilized with 0.1% TritonX‐100 (Beyotime, Shanghai, China) for 30 min. Then cells were blocked with goat serum (Solarbio, Beijing, China) for 15 min, followed by incubation with anti‐NF‐κB p65 (Proteintech Group, IL, USA) at 1:200 dilution in PBS at 4°C overnight. After three times washed with PBS, cells were incubated with Cy3-labeled fluorescent secondary antibody (Beyotime) at 1:200 dilution in PBS and DAPI (Aladdin Regents, Shanghai, China) was used to stain the nucleus. A fluorescence microscope (BX53, Olympus) was then applied to capture images at 400× magnifications.
6
Subcellular Fractionation and Protein Analysis
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Subcellular fractionation was performed with a Nuclear and Cytoplasmic Protein Extraction Kit (Sangon, Shanghai, China), in accordance with the manufacturer’s instructions. Total protein separation and western blotting were performed as described previously [30 (link)]. The following antibodies were used: anti-p-IκBα (Cell Signaling Technology, USA), anti-p-IKKβ (Abcam, Cambridge, MA, USA), anti-β-actin, anti-Lamin B1, anti-TRAF1 and anti-NF-κB p65 (Proteintech, Wuhan, China). All experiments were performed in triplicate.
7
Immunohistochemical Evaluation of Angiogenesis
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IHC was performed following a previously described method [30 (link)]. The following antibodies were used: anti-CD34, anti-VEGF, anti-DNMT1, anti-TRAF1, and anti-NF-κB p65 (Proteintech, Wuhan, China). Staining intensity was scored manually by two independent experienced pathologists as follows: 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The percentage of positive cells was also assessed according to four scores: 1 (0-10%), 2 (11-50%), 3 (51-80%), and 4 (81-100%). The final IHC score was calculated by multiplying the intensity score by the percentage of positive cells. CD34 antibody was used to stain vascular endothelial cells, and then the microvessel density (MVD) was calculated. The field of maximal CD34 expression was found in tumor tissues. Within this field, the area of maximal angiogenesis was selected, and microvessels were counted at 200× magnification. Low MVD was indicated by scores from 0 to 3. High MVD was indicated by scores ≥4 [31 (link)].
8
Immunofluorescence Assay for NF-κB p65 in HCC Cells
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Immunofluorescence assays were performed as described in our previous study [30 (link)]. Treated HCC cells were washed three times with cold phosphate-buffered saline (PBS, Gibco, USA), fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) for 20 min, treated with 0.1% of Triton X-100 (Beyotime, Shanghai, China) for 5 min and blocked in 5% Bovine Serum Albumin (Solarbio, Beijing, China). Then cells were incubated overnight with anti-NF-κB p65 (Proteintech, Wuhan, China) at 4 °C, followed by washing three times with PBS. Next, the cells were incubated with fluorescent Alexa Fluor 594-conjugated goat anti-rabbit IgG (ABclonal, Wuhan, China). Finally, the nuclei were labelled with DAPI (Cell Signaling Technology, USA) for 15 min, and the images of stained cells were captured by the BX41 microscope (Olympus, Japan). All experiments were performed in triplicate.
9
Western Blot Quantification of Key Signaling Proteins
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The protein samples were loaded onto 8% or 10% polyacrylamide gel and transferred to a PVDF membrane by using the semi-dry method. Blocking and antibody reactions were done in blocking buffer containing 5% skim milk (Wako, Osaka, Japan) in Tris-buffered saline containing 0.05% Tween 20 (TBS-T). An anti-MMP9 (Abcam, Cambridge, UK), anti-β-catenin [Cell Signaling Technology (CST), Danvers, MA], anti-phosphorylated STAT3 (CST), anti-NF-κB p65 (CST), anti-STAT3 (Proteintech, Chicago, IL), anti-EpCAM (Abcam), anti-Histone H3 (CST), and HRP-conjugated anti-GAPDH antibody (Wako) were used. The quantitative densitometric analysis was performed using Image J.
10
Quantitative Analysis of Protein Expression in Ear Tissue
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Total proteins from ear tissue and cell samples were extracted with RIPA buffer (Beyotime, China), and were separated with 10 or 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Then, the separated proteins were electrotransferred to PVDF membranes. After blocking the membranes with 5% BSA for 1 h, the membranes were incubated with primary antibody, including anti-MPO (1:500, Proteintech, USA), anti-citH3 (1:1000, Abcam, USA), anti-Nrf2 (1:2000, Proteintech, USA), anti-GAPDH (1:10,000, Proteintech, USA), anti-IL1β (1:1000, Proteintech, USA), anti-IL6 (1:1000, Proteintech, USA), anti-NF-κB p65 (1:2000, Proteintech, USA), anti-NF-κB p65 (phospho, 1:2000, Proteintech, USA), and anti-β-Actin (1:10,000, Proteintech, USA) antibodies at 4 °C for 12 h, and then incubated with the HRP-linked secondary antibody (1:5000, Proteintech, USA). The blots were cut prior to hybridisation with antibodies. The membranes were next visualized with the enhanced chemiluminescence (ECL) assay kit (Yeasen, USA). Image Lab Software was used to detect and quantify the signals of protein bands. The original data of western blot was included in the Supplementary Information file .
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