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. 2008 Jun 30;28(17):5494–5506. doi: 10.1128/MCB.00265-08

FIG. 7.

FIG. 7.

Hsp90β (S226A/S255A) causes post-cytochrome c protection in normal Ba/F3 cell lysates. (A) Control Ba/F3 cells were infected with a retroviral vector encoding Hsp90β (S226E/S255E) or Hsp90β (S226A/S255A) or empty vector. GFP-positive cells were sorted by FACS. Cell lysates were prepared and incubated with 5 ng/μl cytochrome c and 1 mM dATP. Caspase-3 activity was assayed by measuring the cleavage of DEVD-pNA over time (left). Likewise, the lysates were incubated with 1 mM dATP and various concentrations of cytochrome c (Cyt c), and immunoblotting was performed for caspase-9 (C9) and caspase-3 (C3) (right). Procaspase-9/procaspase-3 and cleaved caspase-9/caspase-3 are indicated by arrows and arrowheads, respectively. (B) Ba/F3 cell lysates expressing the empty vector, Hsp90β (S226E/S255E), or Hsp90β (S226A/S255A) were incubated with or without cytochrome c (5 ng/μl) and dATP (1 mM) and loaded onto a Superdex 200 column. Each column fraction was analyzed for Apaf-1 and caspase-9 by immunoblotting.