Journal: Scientific reports

Article Title: Polyphenols journey through blood-brain barrier towards neuronal protection.

doi: 10.1038/s41598-017-11512-6

Figure Lengend Snippet: Figure 4. Effects on neuroinflammation by Cat-sulf and Pyr-sulf. Pro-inflammatory markers were evaluated, namely (a) TNF-α release, (b) intracellular superoxide production, (c) nitric oxide, and (d) CD40 quantified in N9 microglial cells. Cells were pre-incubated for 6 h with each of the bioavailable (poly)phenol metabolite and then challenged with 300ng/mL of LPS. Statistical differences are denoted as ***p < 0.001, **p < 0.01 and *p < 0.05 relatively to lesion (LPS). (e) Microglial NF-κB p65 translocation into the nucleus after 60 minutes of LPS stimulation. Cells were pre-treated with Cat-sulf or Pyr-sulf for 6 h before LPS-stimulation. NF-κB (red); Nuclei (blue) stained with DAPI. Each capture is representative of at least 3 independent biological replicates. Scale bar: 10 µm. (f–i) Microglial NF-κB p65 phosphorylation ratio and IκBα fold change in protein levels. (f) IkBα protein levels along time after LPS stimulation and (g) after 60 min of LPS stimulation with representative western blots. (h) NF-κB activation profile along time after LPS stimulation looking at NF-κB p65 phosphorylation (ser536) ratio along time after LPS stimulation and (i)after 60 min of LPS stimulation with representative western blots. Cells were pre-treated either with Pyr-sulf or Cat-sulf before LPS stimulation. Control cells (white triangles, solid line), LPS-stimulated cells (black triangles, solid line), cells treated with Cat-sulf prior to LPS stimulation (black circles, dashed line), cells treated with Pyr-sulf prior to LPS stimulation (black squares, dotted line). Statistical differences are denoted as *p < 0.05 and **p < 0.01 relatively to lesion (LPS). Western blots were analyzed under the same experimental conditions. Data are presented as the means ± SD, n = 3.

Article Snippet: Briefly, HBMEC coverslips were incubated overnight at 4 °C with primary antibodies anti-P-gp (1:50, Calbiochem), anti-MRP1 (1:100, Millipore) and anti-BCRP (1:100, Millipore) and N9 cells coverslips were incubated overnight at 4 °C with rabbit polyclonal anti-NF-κB p65 (C-20) (1:200, Santa Cruz Biotechnology).

Techniques: Incubation, Translocation Assay, Staining, Phospho-proteomics, Western Blot, Activation Assay, Control

Journal:

Article Title: Activation of NF-?B by Double-Stranded RNA (dsRNA) in the Absence of Protein Kinase R and RNase L Demonstrates the Existence of Two Separate dsRNA-Triggered Antiviral Programs

doi: 10.1128/MCB.21.1.61-72.2001

Figure Lengend Snippet: dsRNA-induced nuclear translocation of NFκB. pkr+/+ (EX12) MEF were treated with Lipofectin alone (A, C, and E) or with pI-pC (10 μg/ml) in the presence of Lipofectin (B, D, and F). At 3 h after the treatment, the cells were fixed and immunostained with an antibody recognizing the p65/RelA subunit of NFκB, as described in Materials and Methods. An irrelevant peptide (representing an epitope corresponding to the C-terminal domain of MEKK1) or a specific blocking peptide were used (as described in Materials and Methods) in panels E and F and in panels C and D, respectively.

Article Snippet: The antibodies against IκB-β (C-20), phospho-(serine-32)–IκB-α (B-9), PKR (M-515 and D-20), and p65/RelA (C-20) and the blocking peptide solutions used in the experiment in Fig. (p65/RelA C-20 peptide and MEKK1 C-22 peptide) were from Santa Cruz Biotechnologies.

Techniques: Translocation Assay, Blocking Assay

Journal:

Article Title: Activation of NF-?B by Double-Stranded RNA (dsRNA) in the Absence of Protein Kinase R and RNase L Demonstrates the Existence of Two Separate dsRNA-Triggered Antiviral Programs

doi: 10.1128/MCB.21.1.61-72.2001

Figure Lengend Snippet: dsRNA-induced nuclear translocation of NFκB in cells deficient in RNase L or both RNase L and PKR. 3T3-like fibroblast cell lines with pkr+/+/rnasel+/+, pkr+/+/rnasel−/−, and pkr−/−/rnasel−/− genotypes were treated with Lipofectin alone (Control) or with pI-pC (10 μg/ml) in the presence of Lipofectin (dsRNA). At 3 h after treatment, the cells were fixed and immunostained with an antibody recognizing the p65/RelA subunit of NFκB as in the experiment in Fig. ​Fig.33.

Article Snippet: The antibodies against IκB-β (C-20), phospho-(serine-32)–IκB-α (B-9), PKR (M-515 and D-20), and p65/RelA (C-20) and the blocking peptide solutions used in the experiment in Fig. (p65/RelA C-20 peptide and MEKK1 C-22 peptide) were from Santa Cruz Biotechnologies.

Techniques: Translocation Assay

Journal:

Article Title: Activation of NF-?B by Double-Stranded RNA (dsRNA) in the Absence of Protein Kinase R and RNase L Demonstrates the Existence of Two Separate dsRNA-Triggered Antiviral Programs

doi: 10.1128/MCB.21.1.61-72.2001

Figure Lengend Snippet: dsRNA-induced nuclear translocation of NFκB independent of the presence or the absence of PKR. pkr+/+(EX2+3), pkr0/0(EX2+3), pkr+/+(EX12), or pkr0/0(EX12) MEF were treated with Lipofectin alone (A, C, E, and G) or with pI-pC (10 μg/ml) in the presence of Lipofectin (B, D, F, and H). At 3 h after the treatment, the cells were fixed and immunostained with an antibody recognizing the p65/RelA subunit of NFκB as in Fig. ​Fig.33.

Article Snippet: The antibodies against IκB-β (C-20), phospho-(serine-32)–IκB-α (B-9), PKR (M-515 and D-20), and p65/RelA (C-20) and the blocking peptide solutions used in the experiment in Fig. (p65/RelA C-20 peptide and MEKK1 C-22 peptide) were from Santa Cruz Biotechnologies.

Techniques: Translocation Assay

Journal:

Article Title: Activation of NF-?B by Double-Stranded RNA (dsRNA) in the Absence of Protein Kinase R and RNase L Demonstrates the Existence of Two Separate dsRNA-Triggered Antiviral Programs

doi: 10.1128/MCB.21.1.61-72.2001

Figure Lengend Snippet: dsRNA- and TNF-α-induced specific DNA-binding activity of NFκB. pkr+/+(EX12) MEF were treated with Lipofectin alone (lanes Co), with pI-pC (10 μg/ml) in the presence of Lipofectin (lanes dsRNA), or with TNF-α (lanes TNF). At 3 h after either Lipofectin or dsRNA treatments or 20 min after the TNF-α treatment, the cells were harvested and nuclear extracts were prepared as described in Materials and Methods. EMSAs were performed as described in Materials and Methods. Where indicated, a 100-fold molar excess of either the specific NF-κB-binding nonlabeled oligonucleotide (specific competitor) or a p53-binding nonlabeled oligonucleotide (nonspecific competitor) was added to the binding-reaction mixtures for 10 min before the addition of the specific 32P-labeled NF-κB-binding oligonucleotide. In the last lane, the undiluted anti-p65/RelA (C-20 from Santa Cruz) antibody was added in 1/10 of the final reaction volume for 10 min before the addition of the specific 32P-labeled NF-κB-binding oligonucleotide. Addition of several irrelevant antibodies did not interfere with the specific binding of NF-κB to DNA, demonstrating the specificity of the anti-p65/RelA antibody-induced supershift (data not shown).

Article Snippet: The antibodies against IκB-β (C-20), phospho-(serine-32)–IκB-α (B-9), PKR (M-515 and D-20), and p65/RelA (C-20) and the blocking peptide solutions used in the experiment in Fig. (p65/RelA C-20 peptide and MEKK1 C-22 peptide) were from Santa Cruz Biotechnologies.

Techniques: Binding Assay, Activity Assay, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Activated CARD11 accelerates germinal center kinetics, promoting mTORC1 and terminal differentiation

doi: 10.1084/jem.20180230

Figure Lengend Snippet: DLBCL B cells expressing aCARD11 exhibit increased pS6 independent of NF-kB signaling. The GCB-DLBCL line, OCI-Ly7, was transduced with lentiviral vectors encoding murine, flag-tagged WT CARD11 or CARD11-L251P, and a cis-linked GFP reporter. (A) Immunoblot showing expression of flag-tagged CARD11 in transduced cell populations. (B) Representative immunoblot of basal signaling in WT or CARD11-L251P cells. (C) Left: Quantification of signaling by using ImageJ of pS6 normalized to total S6 (P = 0.017). Middle: p-p65 normalized to total p65 (P = 0.031). Right: Total IκBα normalized to HSP90 (P = 0.041). Combined data are from three experiments; significance was calculated by Student’s paired t test. Data in A–C are representative of seven biological replicates. (D) NF-κB activity (luciferase) normalized to viability (Cell Titer Glo) after treatment with different doses of IKK-2 inhibitor for 16 h. Area under the curve is significantly different between WT and CARD11-L251P (P = 0.007), calculated by Student’s paired t test. Data represent three independent experiments. (E) Fold change in pS6 median fluorescent intensity of CARD11-L251P expressing OCI-Ly7 cells normalized to untreated cells (P < 0.0001). Data are representative of three independent experiments with three biological replicates. Significance was calculated by one-way ANOVA. (F) Representative histogram overlays of CD98 expression on (left) non-GC B cells (filled histograms: gray, Ctrl; pink, Mb1-aCard11, CD19 + CD95 lo CD38 hi ) and GC B cells (open histograms: black, Ctrl; red, Mb1-aCard11, CD19 + CD95 hi CD38 lo ); (middle) DZ (CD19 + CD95 hi CD38 lo CXCR4 + CD86 − ) and (right) LZ (right, CD19 + CD95 hi CD38 lo CXCR4 − CD86 + ) GC B cells in Ctrl (black) and Mb1-aCard11 (red) mice. (G) Median fluorescent intensity of CD98 on non-GC, GC (P = 0.0008), DZ (P < 0.0001), and LZ cells in Ctrl mice and Mb1-aCard11 mice. (F and G) Data are representative of two independent experiments with five Ctrl mice and seven Mb1-aCard11 mice 5 dpi with SRBCs. Significance was calculated by two-way ANOVA. For summary graphs, lines represent mean ± SEM. For bar graphs, lines represent mean + SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Primary anti–mouse antibodies used for immunoblot analysis included those from Cell Signaling Technology: CARD11 (1D12), HSP90, p-p65 (Ser536, 93H1), p65 (C-20), FOXO1 (D7C1H), p-S6 (Ser235/236), S6 (54D2), and IκBα (5A5); from Santa Cruz Biotechnology: p65; and from Sigma Aldrich: actin (Rb polyclonal).

Techniques: Expressing, Transduction, Western Blot, Activity Assay, Luciferase