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

Additional file 1:

Kinetic analysis of nuclear IκBα translocation. Movie of one CD₄⁺ T lymphocyte transfected with EYFP-IκBα vector photographed before and after treatment with LMB up to 30 minutes. Photographs were taken in vivo by confocal microscopy every minute after adding LMB. This movie corresponds to Figure 3, which has been constructed with some pictures taken at different moments before and after adding LMB. It is an .avi file that can be viewed with Windows Media Player.

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Additional file 2:

Absence of NF-κB binding activity in nuclear protein extracts from unstimulated PHA-treated T cells. (a) Binding of NF-κB in nuclear extracts from PHA-treated T cells to its cognate DNA sequence was analyzed. PBMCs were cultured for 3 days with 5 μg/ml PHA and for the consecutive 9 days with 300 U/ml IL-2. These long-term cultures of PHA-treated T lymphocytes were maintained without supplemental IL-2 18 hours. Three micrograms of nuclear extracts from IL-2 depleted T cells (lane 1) and activated with PMA for 30 min or 5 hours (lanes 2 and 3, respectively) were incubated with an oligonucleotide containing double -κB consensus motif from HIV LTR labeled with [α-³²P]-dCTP. (b) Analysis of the NF-κB complexes composition by supershift assay. Three micrograms of nuclear extracts from PHA-treated T cells activated with PMA for 2 hours were incubated with antibodies against p50/NF-κB1 (lane 3), p65/RelA (lane 4) or c-Rel (lane 5) before the incubation with an oligonucleotide containing double -κB consensus motif from HIV LTR labeled with [α-³²P]-dCTP. Lane 2 shows the specificity of binding of the NF-κB complexes using excess (100×) of unlabelled -κB-motif oligonucleotide as competitor.

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