Figure 5

CCCP promotes reversible Mdm2 recruitment to mitochondria. (A) HeLa cells transfected with the empty vector or HA-Mdm2 were treated with CCCP (10 μM) for indicated times. Cell lysates were collected, and cytosol and mitochondrial fractions were separated, as described in Methods. The lysates and fractions were blotted for HA to detect HA-Mdm2. COX-IV and caspase-3 served as markers for the mitochondrial and cytosolic fractions, respectively. (B) HeLa cells transfected with Mdm2-GFP and mKate-mito were treated with CCCP (10 μM) for indicated times. (C) HeLa cells expressing mKate-mito and Mdm-GFP were treated with 10 μM CCCP for 2 h, whereupon CCCP-containing medium was removed and replaced with CCCP-free medium, in which the cells were incubated ~12 h. Cells were imaged live with Nikon microscope LC2000 and 40X oil objective. In panels (B,C) representative cells co-expressing both proteins (from 2–3 independent experiments performed) are shown.