Journal: Physiological Genomics

Article Title: Superoxide scavenging and Akt inhibition in myocardium ameliorate pressure overload-induced NF-?B activation and cardiac hypertrophy

doi: 10.1152/physiolgenomics.00202.2009

Figure Lengend Snippet: TAB-induced NF-κB activation is attenuated by AdCu/ZnSOD and AdDNAkt. Representative images at day 5 (A) and summary data (B) show the effects of AdLacZ, AdCu/ZnSOD, or AdDNAkt on NF-κB-driven luciferase activity (photon emission) over time in TAB mice compared with sham-operated mice. NF-κB activation was measured by in vivo bioluminescent imaging using Xenogen IVIS-200. In the pseudocolored scale, areas of high photon emission are displayed as red and areas of low photon emission are displayed as blue. Data are means ± SE (n = 4/group) expressed relative to sham-operated mice. *P < 0.05 vs. sham surgery; †P < 0.05 vs. TAB-AdLacZ. DNAkt, dominant-negative form of Akt.

Article Snippet: EMSA was performed with 6 μg of nuclear protein incubated with a γ- 32 P-labeled NF-κB consensus oligonucleotide probe 5′-AGTTGAGGGGACTTTCCCAGGC-3′ (Promega, Madison, WI) as described earlier ( 52 ).

Techniques: Activation Assay, Luciferase, Activity Assay, In Vivo, Imaging, Dominant Negative Mutation

Journal: Current Research in Toxicology

Article Title: Wheatgrass inhibits the lipopolysaccharide-stimulated inflammatory effect in RAW 264.7 macrophages

doi: 10.1016/j.crtox.2021.02.005

Figure Lengend Snippet: Effect of wheatgrass extract on the transactivation of NF-κB promoter in LPS-stimulated RAW 264.7 cells. Raw 264.7 cells were transfected with pNFκB-Luc and β-gal plasmids and then exposed to 1 µg/ml of LPS in the presence or absence of W5 (A) and W7 (B) for 12 h followed by estimation of luciferase activities. Results are mean ± SEM of three independent experiments. * and # indicate p < 0.05 as compared to vehicle-treated control and LPS-stimulated cells, respectively. W5, 5-day old wheatgrass methanol extract; W7, 7-day old wheatgrass methanol extract; LPS, lipopolysaccharide.

Article Snippet: For this assay, RAW 264.7 cells were co-transfected with a p-NF-κB-Luc reporter, containing mammalian NF-κB response element in its promoter region, and β-gal plasmids (both procured from Promega Corporation, Madison, WI, USA), where the latter was used as an internal control.

Techniques: Transfection, Luciferase

Journal: PLoS ONE

Article Title: Lactobacillus acidophilus Alleviates Platelet-Activating Factor-Induced Inflammatory Responses in Human Intestinal Epithelial Cells

doi: 10.1371/journal.pone.0075664

Figure Lengend Snippet: ( A ) p65 levels measured by immunoblotting of nuclear extracts prepared from control or PAF ± LA-CS-treated Caco-2 and NCM460 cells (representative blots from n = 3); ( B ) NF-κB reporter activity, expressed as percent of control, in control or PAF ± LA-CS-treated Caco-2 cells (n = 4, * P <0.05).

Article Snippet: We also measured NF-κB activity by transfecting Caco-2 cells with the NF-κB transcription reporter vector p-NF-κB-Luc (BD Biosciences).

Techniques: Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: Lactobacillus acidophilus Alleviates Platelet-Activating Factor-Induced Inflammatory Responses in Human Intestinal Epithelial Cells

doi: 10.1371/journal.pone.0075664

Figure Lengend Snippet: Model showing mechanisms implicated in alleviating PAF-induced NF-κB activation and IL-8 production in IECs by LA-derived soluble factors.

Article Snippet: We also measured NF-κB activity by transfecting Caco-2 cells with the NF-κB transcription reporter vector p-NF-κB-Luc (BD Biosciences).

Techniques: Activation Assay, Derivative Assay

Journal: PLoS ONE

Article Title: Norisoboldine Suppresses VEGF-Induced Endothelial Cell Migration via the cAMP-PKA-NF-κB/Notch1 Pathway

doi: 10.1371/journal.pone.0081220

Figure Lengend Snippet: ( A ). Effect of NOR on VEGF-induced expressions of p65, p-p65 (ser276), p-p65 (ser536) and PKAc in HUVECs. ( B ) Quantification of expressions of p65, p-p65 (ser276), p-p65 (ser536) and PKAc. ( C ). Effect of NOR on formation of PKAc/p65 complex in VEGF-activated HUVECs. Cells were treated with NOR or Cur in the presence of VEGF for 24 h. Then cell lysates were first immunoprecipitated with PKAc antibody and then immunoblotted with p65 antibody. ( D ). Effect of NOR on the co-localization of PKAc and p65 in HUVECs. Cells were cultured with NOR (10 µM) or Cur (10 µM) in the presence of VEGF (10 ng/ml) for 24 h. Then cells were immunofluorescence stained with PKAc and p65 antibodies. The data were expressed as mean ± SD of three independent experiments. # P <0.05, ## P <0.01 vs . normal group; * P <0.05, ** P <0.01 vs . VEGF group. NOR, norisoboldine; Cur, curcumin.

Article Snippet: SB203580 (a specific inhibitor of p38 MAPK), U0126 (a specific inhibitor of ERK), SP600125 (a specific inhibitor of JNK), triciribine (a specific inhibitor of protein kinase B (AKT/PKB)), chelerythrine (a specific inhibitor of protein kinase C (PKC)), H-89 (a specific inhibitor of PKA), the anti-p-p65 (ser276) antibody, the anti-p-p65 (ser536) antibody, the p-IκBα antibodies, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody, the enhanced chemiluminescent plus reagent kit and peroxidase-conjugated secondary antibodies were purchased from KangChen Bio-tech (Shanghai, China).

Techniques: Immunoprecipitation, Cell Culture, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Norisoboldine Suppresses VEGF-Induced Endothelial Cell Migration via the cAMP-PKA-NF-κB/Notch1 Pathway

doi: 10.1371/journal.pone.0081220

Figure Lengend Snippet: In details, NOR markedly suppressed VEGF-induced intracytoplasmic cAMP production and thereby PKA activation. This action, on one hand, inhibited IκBα degradation and p65/IκBα complex disruption, and then restrained PKAc/p65 complex formation and p65(ser276) phosphorylation to limit transcriptional activation potential of p65; on the other hand, promoted VEGF-induced the release of Notch1 active domain-Cleaved Notch1 to improve expressions of Notch1 targeted genes. NOR, norisoboldine.

Article Snippet: SB203580 (a specific inhibitor of p38 MAPK), U0126 (a specific inhibitor of ERK), SP600125 (a specific inhibitor of JNK), triciribine (a specific inhibitor of protein kinase B (AKT/PKB)), chelerythrine (a specific inhibitor of protein kinase C (PKC)), H-89 (a specific inhibitor of PKA), the anti-p-p65 (ser276) antibody, the anti-p-p65 (ser536) antibody, the p-IκBα antibodies, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody, the enhanced chemiluminescent plus reagent kit and peroxidase-conjugated secondary antibodies were purchased from KangChen Bio-tech (Shanghai, China).

Techniques: Activation Assay