Journal: Foods (Basel, Switzerland)

Article Title: Bixin, a New Atheroprotective Carotenoid Candidate, Prevents oxLDL-Induced Cytotoxicity and Mitochondrial Dysfunction in Macrophages: Involvement of the Nrf2 and NF-κB Pathways.

doi: 10.3390/foods13132002

Figure Lengend Snippet: Figure 6. Bixin and lycopene prevent oxLDL-induced nitrite production (A,B), protein expression of inducible nitric oxide synthase (iNOS), and activation of nuclear factor kappa B (NF-κB) in macrophages cells (C–F). Band quantification and representative Western blotting analysis of iNOS expression (C,D) and of NF-κB cytoplasmic and nuclear expression ((E), bixin; (F), lycopene). β-actin was used to normalize the data for the iNOS and NF-κB cytoplasmic fraction and the proliferative nuclear cell antigen (PCNA) for NF-κB nuclear fraction. Results are the mean ± SEM of three independent experiments. Bars that have no common letters are significantly different (p < 0.05). BIX: bixin; LYC: lycopene; oxLDL: oxidized low-density lipoprotein.

Article Snippet: The membranes were incubated with polyclonal rabbit anti-Nrf2 antibody (1:250; sc-722, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-NF-κB antibody (1:100; sc-166588, Santa Cruz Biotechnology, Dallas, TX, USA), mouse polyclonal anti-β-actin (1:2500; sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA) or mouse monoclonal anti-PCNA (1:200; sc-56, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Activation Assay, Western Blot

Journal: Cancer Biology & Therapy

Article Title: The NFκB inhibitor, SN50, induces differentiation of glioma stem cells and suppresses their oncogenic phenotype

doi: 10.4161/cbt.28158

Figure Lengend Snippet: Figure 3. Effects of silencing of NFκB/p50 expression on differentiation of GSCs. (A) GSC11 cells were dissociated, suspended in serum-free DMEM medium in the absence of B-27, EGF, and β-FGF, and then were infected with an NFκB/p50-targeted shRNA lentiviral particles for 48 h in the presence of polybrene (5 μg/mL). Forty-eight hours later, (A) the cells were observed under a phase contrast microscopy (200× magnification). **P < 0.01, shNFκB/p50 vs. shNT control; (B) cell lysates were prepared and subjected to western blot analysis of CD133, Sox2, MAP-2, and p50. β-actin was used as a loading control.

Article Snippet: GSC11 cells were dissociated, plated into 6-well plates, and then infected with an NFκB/p50-targeted shRNA lentiviral particles (Santa Cruz Biotechnology, Inc.) in the presence of 5 μg/mL of polybrene (Sigma-Aldrich) for 24 h. The cells were then selected with puromycin.

Techniques: Expressing, Infection, shRNA, Microscopy, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Chlamydia abortus Pmp18.1 Induces IL-1β Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways

doi: 10.3389/fcimb.2017.00514

Figure Lengend Snippet: Pmp18.1 activates MyD88, NFκB and Caspase-1 expression in BMDCs. Cell lysates were prepared from BMDCs incubated with Pmp18.1 (10 μg/ml) for 1, 2, or 24 h. Protein extracts (40 μg/well) were separated by SDS-PAGE and detected by immunoblotting analysis incorporating the Pierce™ ECL Western Blotting Substrate using antibodies to the corresponding proteins. The proteins bands were visualized using the ImageQuant LAS-4000 imaging system. Protein levels, normalized to GAPDH, were then quantified using Image J software. The data shows the expression levels and fold induction of TLR4 (A) , MyD88 (B) , NF-κB p50 (C) , and Caspase-1 (D) . Each experiment was repeated at least two times with similar findings. For NF-κB p65 nuclear migration, BMDCs were incubated for 24 h with rPmp18.1 and nuclear NF-κB p65 was detected by confocal microscopy using a FITC conjugated monoclonal antibody to NF-κB p65 (clone D14E12, Cell Signaling Technology). (E) NF-κB p65 unit was stained in green, and nuclei were visualized by DAPI counterstain (blue). Note the diffuse distribution of NF-κB p65 (green) in the nucleus. The statistical significance difference between two groups was evaluated by Student's t -test and between more than two groups by the one-way analysis of variance (ANOVA). Differences were considered to be statistically significant at * p < 0.05.

Article Snippet: Membranes were blocked with 5% non-fat dry milk solution (catalog number 9999) (Cell Signaling Technology, Danvers, MA) at room temperature for 1 h and reacted with the corresponding protein antibodies at 4°C overnight: myeloid differentiation primary response gene 88 (MyD88) (HFL-296, polyclonal; catalog number sc-11356), nuclear factor kappa B (NF-κB) p50 (H-119, polyclonal; catalog number sc-7178), NFκB p65 (F-6, monoclonal; catalog number sc-8008), Caspase-1 (14F468, monoclonal; catalog number sc-56036), GAPDH (FL-335, polyclonal; catalog number sc-25778) (Santa Cruz, Dallas, TX).

Techniques: Expressing, Incubation, SDS Page, Western Blot, Imaging, Software, Migration, Confocal Microscopy, Staining