11 research outputs found

    Diminished Erk activation in RasGRP1<sup>d/d</sup> DP thymocytes.

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    <p>(A–B) Enriched DP thymocytes were stimulated as shown. Lysates were analyzed via SDS-PAGE and immunoblotting for overall tyrosine phosphorylation (pY) and LAT (A) or pErk and Erk2 (B).</p

    Impaired localization of the RasGRP1Δtail construct in Jurkat T cells.

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    <p>Jurkat cells were electroporated with GFP-RasGRP1 or GFP-RasGRP1Δtail plasmids. Live cell imaging was performed on a Zeiss Observer D1 station equipped with a CoolSNAP<sub>HQ</sub> charge-coupled device camera (Roper Scientific) and a high-speed automatic objective stage for multiple Z stack recording. The images were collected with a 40x objective lens and 2.5x camera zoom at 45 second intervals at 37°C. For each cell at each time point, GFP data were collected over 21 continuous vertical Z positions. After the first time frame, OKT3 or PMA were added to the samples. Images were processed by 3D deconvolution using AutoQuant X software (Media Cybernetics). All imaging manipulation and analysis were done using the MetaMorph software suite (Molecular Probes). Data are representative of at least six independent experiments analyzing at least five cells per experiment.</p

    Splenomegaly and autoimmunity in aged RasGRP1<sup>d/d</sup> mice.

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    <p>(A) Spleen weights from 6-month-old mice. (B) Numbers of total, CD4<sup>+</sup>, and CD8<sup>+</sup> splenocytes. (C) Representative FACS plots of CD4 versus CD8 on live splenocytes (top), as well as CD62L and CD44 expression on CD4<sup>+</sup> splenocytes (bottom). (D) TCRβ expression on RasGRP1<sup>−/−</sup> (shaded histogram), RasGRP1<sup>d/d</sup> (solid black line), and RasGRP1<sup>+/+</sup> (dashed black line) splenocytes (left). (E–F) Blood serum levels of IgE, IgG<sub>1</sub> (E), and anti-nuclear antibodies (F) from RasGRP1<sup>−/−</sup> (triangles), RasGRP1<sup>d/d</sup> (squares), and RasGRP1<sup>+/+</sup> (circles) mice. (G) Top panel, auto-antibodies were detected by immunostaining slides with fixed NIH3T3 cells with mouse serum, followed by FITC-conjugated anti-mouse IgG. Bottom panel, immune complex deposition in the glomeruli of mice was visualized by staining kidney cryosections with FITC-conjugated anti-mouse IgG. Imaging was performed using light or fluorescent microscopy at 10x magnification. Two-tailed <i>t</i> test; *, p<0.05; **, p<0.005. Data are representative of at least three independent experiments.</p

    Generation of RasGRP1<sup>d/d</sup> mice.

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    <p>(A) Targeting construct showing insertion of a stop codon into exon 15 of <i>rasgrp1</i>. (B) Real-time PCR analysis of RasGRP1 transcript in DP thymocytes from RasGRP1<sup>+/+</sup> and RasGRP1<sup>d/d</sup> mice. (C) Western blot analysis of RasGRP1 expression in DP thymocyte lysate from RasGRP1<sup>+/+</sup> and RasGRP1<sup>d/d</sup> mice. * indicates two RasGRP1 protein isoforms on the SDS-PAGE.</p

    Aberrant T cell proliferation, activation, and cytokine production in RasGRP1<sup>d/d</sup> mice.

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    <p>(A) Splenocytes were isolated from six to eight-week-old mice and stained with 5 µM CFSE and stimulated as shown. After forty-eight hours, cells were harvested and stained for CD4 and CD8. Histograms shown are gated on CD4<sup>+</sup> cells. Shaded histogram represents RasGRP1<sup>−/−</sup>, solid black line represents RasGRP1<sup>d/d</sup>, and dashed black line represents RasGRP1<sup>+/+</sup> splenocytes. (B) Supernatant from the CFSE culture was collected after 24 hours and used to perform an IL-2 ELISA. (C) Cells were stimulated as in (A) but harvested after twenty-four hours and analyzed by FACS for CD4, CD8, CD69, and CD25 expression. Shaded histogram represents RasGRP1<sup>−/−</sup>, solid black line represents RasGRP1<sup>d/d</sup>, and dashed black line represents RasGRP1<sup>+/+</sup> CD4<sup>+</sup> splenocytes. (D)Splenocytes from six-week-old mice were harvested and stimulated with P+I with Monensin for 4 hours. Left: Representative FACS plots of splenocytes stained with 7AAD, CD4, CD8, and IFN-γ. Right: Quantitative representation of the percent of CD4 and CD8 cells from each genotype that made IFN-γ following <i>in vitro</i> P+I stimulation. Two-tailed <i>t</i> test; *, p<0.05; **, p<0.005. Data are representative of at least three independent experiments.</p

    Comparison of binding affinities for ingenol 3-angelate (I3A) and phorbol 12,13-dibutyrate (PDBu).

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    *<p>Value from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072331#pone.0072331-Kedei1" target="_blank">[9]</a>.</p>†<p>Values represent the mean ± SEM of four independent experiments.</p>‡<p>Value represents the mean ± SEM of three independent experiments.</p

    Activation of RasGRP1 and RasGRP3 by I3A.

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    <p>A. Cultures of Rat2 cells engineered with either empty vector or RasGRP1 cDNA-expressing vector were treated with I3A or PMA for 10 minutes and compared to control cultures using the Ras-GTP pull-down assay. Results are representative of three independent experiments. B. In a single experiment Rat2 cells expressing RasGRP3 cDNA were similarly analyzed. C. Jurkat T cells were analyzed for DAG analogue-induced Ras activation in three independent experiments. D. An equal number of C57Bl/6J (WT, wildtype) and <i>Rasgrp1</i>−/− (KO, knockout) thymocytes were treated with DMSO (negative control), I3A or PMA as indicated for 10 minutes and protein lysates were compared using the phospho-Erk signaling assay. The results are representative of duplicate experiments. Quantitation of the ratios of RasGTP/Ras or p-Erk1/2/Erk1/2 are presented below the individual panels.</p

    Effects of I3A on apoptosis and viable cell accumulation in B-NHL cell lines.

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    <p>TMRE, the percent of cells incapable of assimilating TMRE is shown.</p><p>CaspACE, the percent of cells showing activated caspases is shown.</p><p>TUNEL, the percent of cells showing nuclear DNA fragmentation is shown.</p><p>MTT, the concentration of I3A that caused 50% inhibition of viable cell accumulation was calculated by linear regression.</p>#<p>for TMRE and CaspACE assays cells were treated with 100 nM I3A for 4 days, except RL and Toledo were treated for 24 hr.</p><p>&, for TUNEL assay cells were treated with 100 nM I3A for 7 days, except RL and Toledo were treated for 2 days.</p>*<p>average of two experiments.</p>**<p>average of three experiments.</p>$<p>Rec1 cells do not stain with TMRE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072331#pone.0072331-Stang1" target="_blank">[25]</a>.</p

    Comparison of the potencies of I3A and PMA for biochemical responses at the level of induced protein phosphorylation.

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    <p>A. Ramos cells were treated for 30 minutes with the indicated concentrations of I3A or PMA followed by analysis of cell lysates by immunoblotting. DMSO indicates the vehicle control. Bars indicate quantitation (mean ± SEM) of the results from three independent experiments. A representative immunoblot is illustrated. It should be noted that although the RasGRP3 antibody is not directed against a RasGRP3 phosphorylation site, it consistently yields some increase in signal under conditions of RasGRP3 phosphorylation. B. Ramos cells were treated with 3 nM I3A or PMA, in some cases after pretreatment with Gö6983 (5 µM for 40 min), and analyzed as above. Bars indicate quantitation (mean ± SEM) of the results from two independent experiments. A representative immunoblot is illustrated. An additional experiment performed under similar conditions gave similar results. C. Jurkat T cells were stimulated in a single experiment with I3A for 10 min with or without 10 min pre-incubation with the pan-PKC inhibitor bisindolymaleimide I (4.6 µM), followed by analysis of Ras-GTP, total Ras, pErk1/2 and total Erk1/2 levels.</p

    Quantitative and qualitative changes in Bcl-2 family members induced by I3A in representative B-NHL cells.

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    <p>The indicated cell lines were untreated or treated for 3 days (BL2, UPN1, Z138) or for 24 hours (RL and Toledo). Protein lysates were analyzed by immunoblotting with antibodies to the indicated proteins. Panels A and B were derived from duplicate gels. Erk was used as a loading control.</p
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