nf κb dna binding activity  (Thermo Fisher)

Journal: Theranostics

Article Title: Precision treatment of ventilator-induced lung injury through alveolar epithelial cell targeted lipid nanoparticle delivery

doi: 10.7150/thno.111200

Figure Lengend Snippet: SPFPT@siImp7/PD-LNP inhibits the release of proinflammatory cytokines. AECs were incubated with saline, SPFPT@PD-LNP, SPFPT@siImp7/PD-LNP, and SPFPT@siImp7-LNP for 1 h, and then subjected to cyclic stretch. Static cells served as control. ( A) Representative images of p-p38 expression in AECs under the microscope (Scale bar, 5 μm). Immunostaining was performed with p-p38 (red) and counterstained with DAPI to detect nuclei (blue). (B) Quantification of the nucleo-cytoplasmic distribution of p-p38. (C) Relative NF-κB activity in CS-stimulated AECs incubated with saline, SPFPT@PD-LNP, SPFPT@siImp7/PD-LNP, and SPFPT@siImp7-LNP. (D) Western blotting analysis of p-p38, p38, p-ATF2, p-MK2, p-IκBα, IκBα, p-P65, P65, and GAPDH in AECs. (E) Relative expression of p-p38, p-ATF2, p-MK2, p-IκBα, and p-P65 in AECs measured by Western blotting. (F) AECs were incubated with saline, SPFPT@PD-LNP, SPFPT@siImp7/PD-LNP, SPFPT@siImp7-LNP, and budesonide for 1 h, and then subjected to cyclic stretch. Static cells served as control. The mRNA levels of TNF-α, IL-6, and HMGB1 in AECs were evaluated using qRT-PCR. (G) AECs were incubated with saline, SPFPT@PD-LNP, SPFPT@siImp7/PD-LNP, SPFPT@siImp7-LNP, and budesonide for 1 h, and then subjected to cyclic stretch. Static cells served as control. The protein levels of TNF-α, IL-6, and HMGB1 in the culture supernatant of AECs were evaluated using ELISA. (H) Mice were randomized to the sham-operated group and the VILI group. In the VILI group, mice were intratracheally administered with saline, SPFPT@PD-LNP, SPFPT@siImp7/PD-LNP, SPFPT@siImp7-LNP, and budesonide, respectively. The protein levels of TNF-α, IL-6, and HMGB1 in BALF of mice were evaluated using ELISA. Data are expressed as mean ± SEM from 6 mice per group. Significance: * p < 0.05, ** p < 0.01, ***p < 0.001, ns: no significance.

Article Snippet: The nuclear fraction of cell lysates was obtained using a nuclear protein extraction reagent (Thermo Fisher, Rockford, IL), and the NF-κB (p65) DNA-binding activity in a sample containing 5.0 mg of total protein was determined using the NF-κB (p65) DNA-binding TransAM ELISA kit (Thermo Fisher, Rockford, IL).

Techniques: Incubation, Saline, Control, Expressing, Microscopy, Immunostaining, Activity Assay, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Challenges with Methods for Detecting and Studying the Transcription Factor Nuclear Factor Kappa B (NF-κB) in the Central Nervous System

doi: 10.3390/cells10061335

Figure Lengend Snippet: NF-κB signaling pathway. The transcription factor NF-κB is constitutively expressed in the central nervous system (CNS), where it can be activated by several stimuli, including TNFα, opioids, β-amyloid, and sAPP, to name a few. The NF-κB complex is sequestered in a dimer form (with p65/p50 dimers as the most common composition) in the cytoplasm, where it is bound by IκB. Upon activation by various stimuli, IKK interacts with the inhibitory IκB, resulting in phosphorylation, ubiquitination, and degradation of IκB, rendering the dimer free to translocate to the nucleus. Here, the dimer binds to kappa B binding sites of several gene targets that may be involved in cell survival in neurons or inflammatory pathways in glia. Dimers consisting of p65/p50 tend to be transcriptionally active, whereas p50 homodimers tend to suppress gene transcription. Red text indicates methods appropriate to investigate specific components of NF-κB signaling. BDNF = brain-derived neurotrophic factor; CaMKII = calcium-calmodulin kinase II; ChIP = chromatin immunoprecipitation; ELISA = enzyme-linked immunosorbent assay; EMSA = gel electrophoresis mobility shift assay; IκB = inhibitory κB protein; IKK = inhibitory κB protein kinase; MnSOD = manganese superoxide dismutase; PCR = polymerase chain reaction; PSD-95 = postsynaptic density protein-95; sAPP = secreted amyloid precursor protein; TNFα = tumor necrosis factor alpha.

Article Snippet: On the other hand, EMSA [ ] and commercial DNA binding ELISA (such as TransAM ® NF-κB activation assays from Active Motif ® ) have been widely used to assay NF-κB activation [ , , , ].

Techniques: Activation Assay, Phospho-proteomics, Ubiquitin Proteomics, Binding Assay, Derivative Assay, Chromatin Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Nucleic Acid Electrophoresis, Mobility Shift, Polymerase Chain Reaction

Journal: Cells

Article Title: Challenges with Methods for Detecting and Studying the Transcription Factor Nuclear Factor Kappa B (NF-κB) in the Central Nervous System

doi: 10.3390/cells10061335

Figure Lengend Snippet: Summary of the advantages and disadvantages of the methods used for NF-κB detection.

Article Snippet: On the other hand, EMSA [ ] and commercial DNA binding ELISA (such as TransAM ® NF-κB activation assays from Active Motif ® ) have been widely used to assay NF-κB activation [ , , , ].

Techniques: Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Nucleic Acid Electrophoresis, Mobility Shift, Molecular Weight, Immunohistochemistry, Immunostaining, Amplification, Chromatin Immunoprecipitation, DNA Binding Assay, Immunoprecipitation