American Society of Hematology

Figure 5.

$Figure 5. Effects on various apoptotic regulatory proteins. (A) U937 cells were exposed to 75 nM UCN-01 with or without 400 nM 17-AAG for 30 hours, after which cells were lysed, proteins separated by SDS-PAGE, and Western analysis performed to monitor expression of various proteins. Whole cell lysates were probed for antibodies to PARP, full-length procaspase-9, procaspase-8, and Bid. In addition, cytosolic S-100 fractions were obtained as described in “Materials and methods,” and Western analysis used to monitor cytosolic release of cytochrome c, Smac/DIABLO, and AIF. (B) Cells were treated as described, after which Western analysis was performed to monitor expression of Hsp27, Hsp70, Hsp90, as well as the apoptotic regulatory proteins Bcl-2, Bcl-xL, Mcl-1, XIAP, Bax, and Bak. For each condition, lanes were loaded with 25 μg protein; blots were subsequently stripped and reprobed with antibodies to actin to ensure equivalent loading and transfer. The results of a representative experiment are shown; an additional study yielded equivalent results. CF indicates cleavage fragment.$

Effects on various apoptotic regulatory proteins. (A) U937 cells were exposed to 75 nM UCN-01 with or without 400 nM 17-AAG for 30 hours, after which cells were lysed, proteins separated by SDS-PAGE, and Western analysis performed to monitor expression of various proteins. Whole cell lysates were probed for antibodies to PARP, full-length procaspase-9, procaspase-8, and Bid. In addition, cytosolic S-100 fractions were obtained as described in “Materials and methods,” and Western analysis used to monitor cytosolic release of cytochrome c, Smac/DIABLO, and AIF. (B) Cells were treated as described, after which Western analysis was performed to monitor expression of Hsp27, Hsp70, Hsp90, as well as the apoptotic regulatory proteins Bcl-2, Bcl-xL, Mcl-1, XIAP, Bax, and Bak. For each condition, lanes were loaded with 25 μg protein; blots were subsequently stripped and reprobed with antibodies to actin to ensure equivalent loading and transfer. The results of a representative experiment are shown; an additional study yielded equivalent results. CF indicates cleavage fragment.

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