Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-0

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>antibodies against IκBα and p65/RelA. A secondary antibody conjugated with Texas Red (Molecular Probes) was used. Images were taken by confocal microscopy

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-3

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>�B/IκBα complexes in the nucleus after treatment with LMB or PMA. Ten micrograms of cytosolic and nuclear extracts from CDT cells treated with either PMA or LMB during 4 and 6 hours respectively were analyzed by Western Blot using antibodies against p65/RelA and IκBα. Immunoprecipitation assays were performed using 100 μg of these cytosolic and nuclear extracts, which were incubated with 5 μg of an antibody against p65/RelA conjugated with agarose. IκBα and p65/RelA complexes were characterized by immunoblotting. (b) Contamination with cytosolic proteins during nuclear protein extraction or accumulation of cytosolic proteins in the nucleus after treatment with LMB was assessed by Western Blot using an antibody against both p105 and p50/NF-κB1 proteins. (c) Analysis of NF-κB DNA-binding activity in CDT cells treated with either PMA or LMB. Three micrograms of nuclear extract were incubated with an oligonucleotide containing the double consensus motif κB present in the HIV LTR labeled with [α-P]-dCTP. Protein extracts were obtained from CDT cells after treatment with either LMB or PMA for 6 and 4 hours respectively. (d) Analysis of IκBα pool dependence on protein synthesis. Ten micrograms of nuclear extracts from CDT cells incubated with 20 nM LMB for 4 hours and 10 μg/ml CHX and/or 25 ng/ml PMA for 4 hours,30 min and 2 hours, respectively, were analyzed by Western Blot

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-1

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>ctors per million of cells. LMB was added immediately after transfection. After 18–24 hours of incubation, cells were analyzed by confocal microscopy. pEYFP-C1 vector was used as control of unspecific distribution. (b) Resting purified CDT lymphocytes were transiently transfected with the control plasmid pcDNA3.1 by using an Amaxa nucleofector and a classical electroporator (Equibio). As occurs in untransfected resting T cells (lane 1), NF-κB was not induced in resting CDT lymphocytes after electroporation (lanes 3 and 4). As a positive control, NF-κB (p50/p65) binding was induced in these cells by PMA activation (lane 2)

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-4

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>expression vectors or (b) pcDNA3.1 and/or CMV-Tat and/or CMV-IκBα expression vectors, as indicated. Cells were treated with LMB immediately after transfection and/or with PMA two hours after transfection, as indicated. Luciferase activity was measured 18 hours after transfection. Numbers on the top of the bars represent fold transcriptional activity relative to unstimulated T cells transfected with pcDNA3.1

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-8

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>t with LMB up to 30 minutes. Photographs were taken by confocal microscopy every minute after adding LMB

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-7

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>ctors per million of cells. LMB was added immediately after transfection. After 18–24 hours of incubation, cells were analyzed by confocal microscopy. pEYFP-C1 vector was used as control of unspecific distribution. (b) Resting purified CDT lymphocytes were transiently transfected with the control plasmid pcDNA3.1 by using an Amaxa nucleofector and a classical electroporator (Equibio). As occurs in untransfected resting T cells (lane 1), NF-κB was not induced in resting CDT lymphocytes after electroporation (lanes 3 and 4). As a positive control, NF-κB (p50/p65) binding was induced in these cells by PMA activation (lane 2)

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-2

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>t with LMB up to 30 minutes. Photographs were taken by confocal microscopy every minute after adding LMB

Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression-5

<p><b>Copyright information:</b></p><p>Taken from "Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression"</p><p>http://www.retrovirology.com/content/4/1/56</p><p>Retrovirology 2007;4():56-56.</p><p>Published online 8 Aug 2007</p><p>PMCID:PMC1988826.</p><p></p>together with CMV-IκBα or pcDNA3.1 as negative control, and then activated with anti-CDand IL-2, PHA and IL-2, or maintained in the absence of activation. Viral replication was determined by quantification of HIV p24-gag antigen in culture supernatants (a) after 5 days of transfection or (b) after 7 days of transfection. Numbers on the top of the bars represent fold HIV-replication relative to unstimulated T cells transfected with pcDNA3.1. Differences in p24-gag production were significant for resting and anti-CD-activated T cells (p < 0.05) and a trend towards statistical significance was found in PHA-activated T cells (p = 0.081)

(A–E) Western analysis of signaling proteins in J77-GFP or dynamitin-disrupted cells upon stimulation with SEE for the times indicated

Whole lysates were processed for phosphorylated and total protein expression as follows: (A) ZAP70 pY315 and total ZAP70; (B) LAT pY132, pY171, pY191, pY226, and total LAT; (C) Vav1 pY174 and total Vav1; (D) PKCθ pT538 and total PKCθ; and (E) phosphorylated and total Erk1/2, followed by the corresponding HRP-conjugated secondary antibodies. One out of four representative experiments is shown. Arrows indicate target band sizes. (F–H) Conjugates formed and stained for proteins involved in IS formation and maturation (red in merged images, Alexa 568–labeled anti–mouse secondary antibody): LAT (F; arrowheads indicate the fraction of LAT associated with the nontranslocated MTOC in the dynamitin-disrupted clones and not delivered to the nascent IS), pY174Vav1 (G), and PKCθ (H). Arrows indicate the IS area. R, SEE-Raji APC (CMAC-labeled, blue). Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "MTOC translocation modulates IS formation and controls sustained T cell signaling"</p><p></p><p>The Journal of Cell Biology 2008;182(5):951-962.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528574.</p><p></p

(A) CD69 induction was detected by flow cytometry in J77 Jurkat clones overexpressing GFP or p50-dynamitin-GFP (c) and conjugated with unprimed or SEE-pulsed Raji APC for 16 h (percentage of cells is shown)

Three experiments were performed in triplicate. (B and C) IL-2 was detected by ELISA in culture supernatants (B) or cell lysates (C) from the cultures in A. Three experiments were performed in triplicate. (D) IL-2 detected by ELISA in cell supernatants from DHC-silenced J77 cells (siDHC, closed bars) and controls (siNeg, open bars) conjugated with SEE-pulsed Raji APC with the indicated doses (μg/ml). Three experiments were performed in triplicate. Data are means ± SD. *, P < 0.05; **, P < 0.001 (Student's test).<p><b>Copyright information:</b></p><p>Taken from "MTOC translocation modulates IS formation and controls sustained T cell signaling"</p><p></p><p>The Journal of Cell Biology 2008;182(5):951-962.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528574.</p><p></p