Figure 6
Effect of a combination of nutlin-3 and ABT-737 in the EBV (+) cell lines. BL2B95 EBV (+) cells were or were not subjected to prior treatment with 10 μM ABT-737 for 1 h, and were then treated with nutlin-3 for 7 h. (a) The levels of Bcl-2 and Bax proteins were assessed by western blot analysis. (b) Lysates were subjected to immunoprecipitation with agarose-conjugated anti-Bax pAb (IP) or agarose-conjugated control rabbit IgG (IgG control) and then subjected to western blotting with an anti-Bcl-2 mAb or an anti-Bax pAb. As a control for protein levels before IP, a portion of cell lysate (Input) corresponding to 15% of the input for IP was also included in the western blot. All results are representative of three independent experiments. (c) BL2/B95 EBV (+) cells were or were not subjected to prior treatment with 10 μM ABT-737 for 1 h and were then treated with nutlin-3 for the indicated periods of time. Cytosolic and mitochondrial fractions were analyzed by western blotting with an anti-Bax pAb. Vinculin and Bcl-2 were used as cytosolic and mitochondrial markers, respectively. Fold-change values versus the untreated control (0 h), after normalization with respect to the levels of vinculin or Bcl-2 protein, are shown under the blots. The results shown are representative of three independent experiments. (d) BL2/B95 cells were or were not subjected to prior treatment with ABT-737 for 1 h and were then left untreated or treated with nutlin-3 for 24 h. Cells were fixed in 0.25% paraformaldehyde and labeled with a conformation-specific 6A7 Bax antibody, which recognizes only the active conformation of the protein and Alexa 488-conjugated goat anti-mouse IgG. Cells were then analyzed with a FACSCalibur flow cytometer
