Hsp70 Prevents Bax Membrane Insertion

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Hsp70 Prevents Bax Membrane Insertion

• Stankiewicz et al. JBC (2005) 280 38729-38729.

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Stankiewicz et al. JBC (2005) 280 38729-38729.
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FIGURE 1. Hsp70 prevents Bax translocation in
heat-shocked cells. A, Hsp70 prevents Bax
translocation, and this requires the chaperone
function of Hsp70. Immunofluorescence analysis
conducted with a conformation-specific anti-Bax
antibody in non-induced (OFF) and induced (ON)
cells expressing either Hsp70 or Hsp70 lacking
its ATPase domain. Heat-stressed cells were
exposed to 43 C for 60 min and then returned to
37 C for 6 h. Nuclei are stained with
4,6-diamidino-2-phenylindole dihydrochloride. B,
Bax-positive cells were counted in control
cultures and in cultures that were heat-shocked
and either analyzed immediately (HS/0) or after a
6-h incubation at 37 C (HS/6). At least 400
cells were counted for each sample.
Stankiewicz et al. JBC (2005) 280 38729-38729.
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Stankiewicz et al. JBC (2005) 280 38729-38729.
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C, heat shock does not alter Bax protein levels.
Cells were exposed to 43 C for either 60 or 90
min and then returned to 37 C for 6 h. Western
blotting was performed using the indicated
antibodies (p17 represents the processed fragment
detected with the anti-caspase 3 antibody). D,
association of Bax with intracellular membranes
in heat-shocked cells. Soluble (S), membrane (M),
and pellet (P) fractions were obtained by
differential detergent lysis and centrifugation.
Bax is primarily soluble in control cells (C) but
becomes membrane-associated after exposure to
heat shock (60 or 90 min at 43 C followed by 6 h
at 37 C). This translocation from the cytosol to
membrane fraction is inhibited in the
Hsp70-expressing cells (ON). Cytochrome oxidase
subunit II (COXII) reactivity demonstrates the
purity of the membrane and soluble fractions.
Stankiewicz et al. JBC (2005) 280 38729-38729.