Figure 4

Analysis of nuclear NF-κB/IκBα complexes in CD ₄ ⁺ T cells and IκBα pool dependence on de novo protein synthesis. (a) Analysis of subcellular distribution of p65/RelA and IκBα in CD₄⁺ T cells and presence of NF-κB/IκBα complexes in the nucleus after treatment with LMB or PMA. Ten micrograms of cytosolic and nuclear extracts from CD₄⁺ T 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 CD₄ ⁺T 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-³²P]-dCTP. Protein extracts were obtained from CD₄⁺ T cells after treatment with either LMB or PMA for 6 and 4 hours respectively. (d) Analysis of IκBα pool dependence on de novo protein synthesis. Ten micrograms of nuclear extracts from CD₄⁺ T 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.