American Society of Hematology

Figure 3

$Figure 3. Exposure to perifosine/PD184352 results in Bak and Bax conformational change, whereas knockdown of these molecules markedly attenuates apoptosis. (A) U937 cells were exposed to PD184352 (10μM) and perifosine (2.5μM) alone or in combination for 16 hours, after which cells were lysed in buffer containing 1% CHAPS; conformationally changed Bak and Bax protein were immunoprecipitated using anti-Bak Ab1 and anti-Bax 6A7 antibodies, respectively, and subjected to Western blot analysis using polyclonal Bak and Bax antibodies (top panel). Alternatively, cytosolic and membrane fractions were separated, as indicated in “Subcellular fractionation,” and subjected to Western blot analysis (bottom panels). (B) U937 cells were exposed to PD184352 and perifosine alone or in combination for 6 or 16 hours, after which protein lysates were prepared and subjected to Western blot analysis. (C) U937 cells were treated and lysed as in panel A and subjected to immunoprecipitation using anti-Bak antibodies. The immunoprecipitates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted with polyclonal Bax antibodies. Input lysates were subjected to Western blot and probed with Bak antibodies. (D) U937 cells stably transfected with shGFP, Bax (shBax), or Bak (shBak) were treated with perifosine and PD184352, or with VP16 (2.5μM) for 24 hours, after which protein lysates were prepared and subjected to Western blot analysis. Alternatively, the extent of apoptosis was determined using annexin V staining (E). Values represent the means for 3 separate experiments ± SD. *Significantly lower than values obtained for shGFP cells (P < .01).$

Exposure to perifosine/PD184352 results in Bak and Bax conformational change, whereas knockdown of these molecules markedly attenuates apoptosis. (A) U937 cells were exposed to PD184352 (10μM) and perifosine (2.5μM) alone or in combination for 16 hours, after which cells were lysed in buffer containing 1% CHAPS; conformationally changed Bak and Bax protein were immunoprecipitated using anti-Bak Ab1 and anti-Bax 6A7 antibodies, respectively, and subjected to Western blot analysis using polyclonal Bak and Bax antibodies (top panel). Alternatively, cytosolic and membrane fractions were separated, as indicated in “Subcellular fractionation,” and subjected to Western blot analysis (bottom panels). (B) U937 cells were exposed to PD184352 and perifosine alone or in combination for 6 or 16 hours, after which protein lysates were prepared and subjected to Western blot analysis. (C) U937 cells were treated and lysed as in panel A and subjected to immunoprecipitation using anti-Bak antibodies. The immunoprecipitates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted with polyclonal Bax antibodies. Input lysates were subjected to Western blot and probed with Bak antibodies. (D) U937 cells stably transfected with shGFP, Bax (shBax), or Bak (shBak) were treated with perifosine and PD184352, or with VP16 (2.5μM) for 24 hours, after which protein lysates were prepared and subjected to Western blot analysis. Alternatively, the extent of apoptosis was determined using annexin V staining (E). Values represent the means for 3 separate experiments ± SD. *Significantly lower than values obtained for shGFP cells (P < .01).

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