Fig. 6.

N-terminal epitope exposure of endogenous Bax in membrane complexes during anoikis. a) Membrane fractions isolated from adherent cells and cells detached for various times were extracted separated by BN-PAGE before immunoblotting with mAb 5B7. 5B7 predominantly detected a 400kDa species in the membrane fraction of detached cells, but 2D-analysis suggests that this is non-specific. Equal amounts of the membrane fraction from cells detached for 1 hour were treated with octylglucoside, Triton X100 or SDS before BN-PAGE and immunoblotting with 5B7. All three detergents resulted in an increase in immunoreactive Bax. b) Cytosolic and membrane fractions from adherent and detached FSK-7 cells were separated by BN-PAGE and transferred to PVDF. The native membrane was immunoblotted with mAb 5B7. c) The same immunoblot as shown in panel B was denatured and reprobed with mAb 5B7 a second time. Bax was now detected as a monomer and in the 200kDa complex. The area of the immunoblot within the dotted line is shown at a lower exposure in the adjacent panel to allow comparison. Note that the distribution of Bax seen with 5B7 in the denatured blot is similar to that shown earlier with a polyclonal anti-Bax antibody in a native blot (Fig. 2a). All blots are representative examples of at least three experiments.