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: Basic & clinical pharmacology & toxicology

Article Title: Effects of insulin-like growth factor-1 on rotenone-induced apoptosis in human lymphocyte cells.

doi: 10.1111/j.1742-7843.2009.00472.x

Figure Lengend Snippet: Fig. 4. Rotenone (ROT) induced simultaneous NF-jB, p53 and c-Jun transcription factor activation in lymphocytes. Peripheral blood lymphocytes cells were left untreated (A–C), exposed to 250 lM rotenone (D–F), 250 nM IGF-1 (G–I), and to 250 nM IGF-1 + 250 lM rotenone (J–L) for 24 hr. After the incubation period, cells were stained with anti-NF-jB-p65 (A,D,G,J), anti-p53 (B,E,H,K) and anti-c-Jun (C,F,I,L) antibodies according to the procedure described in Materials and Methods. Notice that NF-jB, p53 and c-Jun positive-nuclei (dark brown) reflect their nuclear translocation ⁄ activation. Magnification 400· (A–L).

Article Snippet: The supplier’s protocol (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; goat ABC staining System: cat # sc-2023) was followed for the immunocytochemistry using primary goat polyclonal antibodies NF-jB p65 (C-20)-G (Santa Cruz 2009 The Authors Journal compilation 2009 Nordic Pharmacological Society.

Techniques: Activation Assay, Incubation, Staining, Translocation Assay

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