(A) Lack of mitochondrial localization by ΔN30MEF2D. Western blotting showed the presence of overexpressed ΔN30MEF2D in cytoplasmic and nuclear fractions, but not in the mitochondrial fraction, of SN4741 cells (n = 3). VDAC, PARP, and c-Raf are mitochondrial, nuclear, and cytoplasmic markers, respectively. Control indicates the control vector group. (B) Immunocytochemistry analysis of mitochondrial localization of transfected MEF2D-Flag. Overexpressed ΔN30MEF2D did not colocalize with MitoTracker in SN4741 cells (n = 50 cells; **P < 0.01). Experiments were repeated 4 times. Scale bars: 15 μm. (C and D) Requirement of mtHsp70 for mitochondrial targeting of MEF2D (n = 4; **P < 0.01). Control indicates untreated. Knocking down mouse mtHsp70 by siRNA reduced MEF2D level in purified mitochondria from SN4741 cells (C). Knocking down mouse mtHsp70 by siRNA did not reduce whole cell MEF2D level in SN4741 cells (D). MnSOD is a known mtHsp70-imported mitochondrial matrix protein.