Figure 2. BK_Ca interacts with Tom22 in HEK293T cells.

Cells were co-transfected with BK_Ca, BK_Ca-DEC and Tom22 in different combinations (+, present; -, absent). Co-immunoprecipitated products are highlighted with squares. (A) Immunoblot demonstrating that BK_Ca and BK_Ca-DEC pull down Tom22. BK_Ca constructs were immunoprecipitated with anti-HA pAb and detected with anti-HA mAb (arrows) and co-immunoprecipitated Tom22 was detected with anti-c-Myc mAb (arrowhead, squares). Primary antibodies were incubated at the same time. (B) Immunoblot showing proper expression of BK_Ca constructs (arrows) and Tom22 (arrowhead) in the input lysates. (C) BK_Ca and BK_Ca-DEC efficiency in pulling down Tom22 is similar (n=3 each). Co-IP efficiency was determined by first subtracting background signals, which in this case were negligible, and calculating the ratio of the band intensities of co-immunoprecipitated Tom22 (i.e. A, squares) to corresponding immunoprecipitated BK_Ca constructs (i.e.. A, arrows), and normalized to Tom22 intensity in the corresponding input lysates (i.e. B, arrowhead). In this and following figures, image analysis was performed with MetaMorph Image Analysis Software (Molecular Devices).