Figure 9.

Increase of HIV-1 replication in resting PBLs after over-expression of ΔNH₂p65-tag. (a) Resting PBLs were transfected with pNL4.3-Renilla (a) or pNL4.3-wt (b) vectors together with pCMV-p65wt-tag and pCMV-ΔNH₂p65-tag expression vectors, separately or in ratio 2:1, 1:1, and 1:4. Cells were maintained in culture in the absence of activation for 72 hours and then HIV-1 replication was assessed by quantification of Renilla RLUs in whole protein extracts or HIV-1 p24-gag antigen in culture supernatants. Internal control of transfection was carried out by co-transfection of the pSV-β-Galactosidase vector. Data correspond to the mean of three different experiments and lines on the top of the bars represent the standard deviation. (b) Two hundred micrograms of cytosolic and nuclear extracts from resting PBLs transfected with the control plasmid pCMV-Tag1 or pCMV-p65wt-tag and pCMV-ΔNH₂p65-tag expression vectors were analyzed by immunoprecipitation with the anti-FLAG tag M2 mAb and subsequent immunoblotting with an antibody against the carboxy terminus of p65/RelA.