Figure 4

Caspase-3 activity is related to the cleavage of p65/RelA in non-apoptotic PBLs after PMA- or PHA-activation. Human PBLs were cultured in the presence of PMA or PHA for 4 days and protein extracts were then analyzed by immunoblotting using an antibody against full-length precursor of caspase-3 (32 kDa), p17 and p20 subunits (a), and against the carboxy-terminus of p65/RelA and NF-κB1/p50 (b). (c) Caspase-3 activity was measured in PBLs after treatment with PMA or PHA for 4 days and (d) viability of human PBLs cultured in the presence of PMA or PHA for 1 to 4 days was measured in comparison with PBLs treated with DEM at 0,4 mM. Data correspond to the mean of three different experiments and lines on the top of the bars represent the standard deviation. (e) Jurkat cells were transiently transfected with either pCMV-p65wt-tag or pCMV-p65 D94E;D97E-tag expression vectors. Cells were then activated with PMA immediately after transfection (for 18 hours, lanes 4 and 8), or maintained for 14 hours without previous stimulus and then treated with PMA for 1 hour (lanes 2 and 6) or 4 hours (lanes 3 and 7). Analysis of protein expression was performed by immunoblotting using an antibody against the carboxy-terminus of p65/RelA. Gel bands were quantified by densitometry and background noise was subtracted from the images. Relative ratio of optical density units was calculated regarding to the gel band with less optical density.