Figure 1. MitoTimer localizes to mitochondria. (A) HEK 293 cells were transfected with pTRE-MitoTimer (MT), exposed to Dox (+) or vehicle (−) for 24 h, then subjected to subcellular fractionation to obtain mitochondria (lanes 1–3) and cytosol (lanes 5–7). As a positive control for the DsRed antibody, which recognizes DsRed as well as MitoTimer, a dish of cells were transfected with mitochondrial-targeted DsRed (DsR, lanes 3 and 7). Blots were probed with antibody to COX IV (mito marker) and RHO (cytosol marker). (B) Deconvolved fluorescence image (merged red and green channels) of a transfected and Dox-induced cell showing subcellular localization of MitoTimer. Scale bar: 10 µm. (C) Cells were transfected with MitoTimer and induced with Dox for 24 h, then disrupted to obtain whole cell lysate (WCL), cytosol (Cyto), and mitochondria, which were further disrupted to obtain intermembrane space (IMS), matrix (Mtx), inner membrane (IM), and outer membrane (OM). MitoTimer distribution was detected with antibody to DsRed. RHO was used as a cytosol marker, ACO2 as a matrix marker, and COX IV as an inner membrane marker.