Figure 4. Oligomerization of Bcl-2 and Bax in liposomal membrane.

A, homo-association of His₆-Bcl-2ΔTM in solution (pH 7.4) was detected by crosslinking with BMH. Data shown is a representative Coomassie-stained SDS-PAGE gel from three independent experiments. (●), Bcl-2 monomer; arrow, crosslinked Bcl-2 dimer. B, homo-association of His₆-Bcl-2ΔTM in the liposomal membrane was monitored by BMH crosslinking after the protein was incubated with the liposome at pH 5.0, and the membrane-bound protein was purified using sucrose float-up centrifugation. Data shown is a representative from two independent experiments. C, oligomerization of 50 or 800 nM His₆-Bcl-2ΔTM in the membrane was determined by gel filtration chromatography after incubating the protein at pH 7.4 with 12.5 μM Ni²⁺-liposome in the absence or presence of 5 nM tBid. The proteins in the eluted fractions were analyzed by SDS-PAGE and immunoblotting with a Bcl-2-specific antibody. The elution positions of protein standards are indicated on the top of the plot with M_r. D, oligomerization of 200 nM Bax in the membrane in the absence or presence of 20 nM tBid was determined similarly, except that a Bax-specific antibody was used in the immunoblotting.