Immunoprecipitation and immunodepletion for cytoplasmic and mitochondrial extracts.
(A) Coimmunoprecipitation of BAD:BCL-2 and BAD:BCL-XL in BCR/ABL-expressing cells. HA-BAD immunoprecipitates (IP) from cytoplasmic and mitochondrial extracts of WT BAD- and DM BAD-infected BCR/ABL-expressing cells were blotted with anti–BCL-XL, anti-BCL-2, and anti-HA antibodies. Preclearings were used as controls. Lane HM shows BCL-XL,BCL-2, and BAD expression in mitochondrial extracts from BCR/ABL/WT BAD-expressing cells. In the upper panel, samples were electrophoresed in the presence of β-mercaptoethanol as reducing agent. (B) BCL-2 and BCL-XL levels in HA-BAD-immunodepleted mitochondrial extracts. Western blot shows levels of BCL-2 and BCL-XL in mitochondrial extracts of WTHA-BAD– (lanes 1 and 3) or DMHA-BAD– (lanes 2 and 4) expressing cells before (lanes 1 and 2) or after (lanes 3 and 4) immunodepletion with the anti-HA antibody. Levels of HA-BAD and COX IV were measured as control of immunodepletion efficiency and of equal loading, respectively.
