CA4 is purchased from Santacruz. All other chemical and reagents were obtained from Fisher Scientific Company. Antibodies to cleaved PARP, cleaved caspase, cyclin B1, aurora kinase A, CDK1 and CDC25C, COX-2, NF-κβ, E-Cadherin and Snail proteins were purchased from Cell Signal Technology.
Nf κβ
Manufactured by Cell Signaling Technology
NF-κβ is a protein complex that acts as a transcription factor, regulating the expression of genes involved in immune response, inflammation, and cell growth. It plays a crucial role in cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Cox 2, by Cell Signaling Technology (2 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (2 mentions)
Vimentin, by Abcam (2 mentions)
N cadherin, by Abcam (2 mentions)
Gsk 3β, by Abcam (2 mentions)
Cdc25c, by Cell Signaling Technology (1 mentions)
Cyclin b1, by Cell Signaling Technology (1 mentions)
Aurora kinase a, by Cell Signaling Technology (1 mentions)
Snail, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Tnf α, by Cell Signaling Technology (1 mentions)
Myd88, by Cell Signaling Technology (1 mentions)
Timp 2, by Cell Signaling Technology (1 mentions)
Traf6, by Cell Signaling Technology (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
P pdgfrα, by Cell Signaling Technology (1 mentions)
6 protocols using nf κβ
1
Apoptosis and Cell Cycle Regulation
2
Aortic Protein Extraction and Western Blot
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The aorta was homogenized in lysis buffer (Cell Signaling technology, Danvers, MA, USA) with PMSF at 4℃, and centrifuged (10,000 × g). After this, the supernatants were centrifuged at 13,000 × g. The protein content of each of the samples was determined using Bradford's method, with bovine serum albumin as a standard. The protein samples (35 µg) were boiled in 5× sample buffer followed by polyacrylamide resolving gel and stacking gel, and then transferred overnight to a nitrocellulose membrane at 15 volts. The membranes were washed, blocked, and incubated with Western blotting to detect NF-κβ, TNF-α, IL-6, Cox-2 (Cell Signaling, Danvers, MA, State, 1:1000), and eNOS (1:2000) (BD Biosciences, Bedford, MA) for overnight at -4℃. Secondary antibodies were incubated with horseradish peroxidase-conjugated secondary antibody for 1hr (1:3000, Santa Cruz Biotechnology) at room temperature. The membranes were washed and visualized by autoradiography after development with ECL (Amersham Life Sciences, Arlington Heights, IL, USA). Densitometry was performed with gel documentation (Gel Doc 2000, Quantity One Bio Red, Hercules, CA, USA).
3
Liver Protein Analysis by Western Blot
4
Multiplex Analysis of Neuroinflammation and Neurodegeneration
5
Immunofluorescence Analysis of EMT Markers
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The cells cultured on glass coverslips were fixed at the indicated time points after the treatments (see supplementary Fig. 1 ). Fixation was performed with 3.7% formaldehyde in PBS and permeabilization with 0.5% Triton-X-100 (Merck) in PBS, each step for 30 min at 4 °C. After fixation, the samples were blocked with 2% bovine serum albumin (BSA, Sigma) for 30 min at 37 °C and immediately incubated for 1 h at 37 °C with the primary antibodies (E-cadherin, β-catenin (BD Transduction Laboratories), N-cadherin, Vimentin, GSK3β (Abcam), NF-κβ (Cell Signaling) and Snail (Invitrogen)). Then, cells were washed with PBS and incubated with the corresponding secondary antibodies. Nuclear counterstaining was performed with 0.2 μg/ml Höechst-33258 (H-33528) in distilled water, for 5 min at RT. The samples were then washed and mounted with Prolong™ Gold reagent (Life Technologies). Mitotic index was determined by counting cells in division divided by total cells.
6
Western Blot Analysis of Cell Lysates
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For Western blot analysis, cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 10 mM Tris–HCl pH 7.2, 5 mM EDTA), containing the appropriate concentration of Phosphatase Cocktail and Protease Inhibitor Cocktail (Sigma-Aldrich). Protein concentration was measured by the BCA Protein Assay Kit (Termo Scientific Pierce, Rockford, IL, USA). The proteins were electrophoresed and blotted on Immobilon-P PVDF membranes (Millipore Co., MA, USA). Membranes were blocked in PBS-tween 0.1% with 5% non-fat dried milk for 1 h at 25 °C and then incubated with the first antibody overnight at 4 °C (E-cadherin, β-catenin (BD Transduction Laboratories), N-cadherin, Vimentin, GSK3β, GAPDH (Abcam) and NF-κβ (Cell Signaling)). After washing with PBS-tween 0.1%, membranes were subjected to the peroxidase-conjugated secondary antibody and developed by chemiluminescence (ECL, Amersham Pharmacia Biotech, Little Chalfont, UK) employing the high-definition system ChemiDocTR XRS + (Bio-Rad Laboratories, Hercules, CA, USA). The bands corresponding to the different proteins were digitalized employing the Image Lab version 3.0.1 (Bio-Rad Laboratories).
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