FIG. 6.
Subcellular localization and homodimer formation of BAX in Jurkat cells. (A) Subcellular localization of BAX wt and mIII-1 in Jurkat cells. Jt-Baxwt and Jt-HABaxmIII-1 cells before and 24 h after doxycycline (Dox) treatment were suspended in isotonic buffer, homogenized with a polytron homogenizer, and separated into soluble fraction (S100), LM fraction, HM fraction, and low-speed pellet (P1) by differential centrifugation. The fractions were analyzed by Western blot hybridization with anti-BAX (N20), anti-HA (12CA5), or anti-cytochrome c (Cyto c) Abs. (B) HMs prepared from Jurkat cells after 24 h of treatment with 1 μg of doxycycline per ml were incubated in isotonic buffer and treated with membrane-impermeable BS3 or membrane-permeable DSS cross-linkers or with dimethyl sulfoxide (DMSO) as a control. After treatment, membranes were lysed, cleared by centrifugation, and separated by SDS-PAGE, followed by Western blot hybridization with anti-BAX or anti-HA Abs. At the left are molecular size markers (in kilodaltons).