(A and B) Dually labeled immunoblots of BKCa (A, top) or BKCa-DEC (B, top) co-expressed with Tom22 (bottom) in different cellular fractions: CL, whole cell lysate; Cmito, “crude” mitochondria; Pmito, Percoll-purified mitochondria; Cmem, “crude” membranes; cytosol, cytosolic fraction. BKCa and Tom22 images were separated for clarity. In panel (A), BKCa and Tom22 blots are from different experiments. BKCa constructs were recognized with anti-HA mAb (upper panels); Tom22 was recognized with anti-c-Myc pAb (lower panels). (C-E) Mean values of BKCa, BKCa-DEC and Tom22 relative abundance in different cellular fractions show that BKCa, BKCa-DEC and Tom22 were imported into “crude” and purified mitochondrial fractions, though not enriched. Values for Tom22 were pooled from both BKCa and BKCa-DEC experiments. *, p<0.05 compared to CL. **, p<0.01 compared to CL. n=3 independent experiments.