Fig. 5
MnSOD and Tom20 physically associate with each other, and inhibition of Tom20 reduces monensin-induced increase of mitochondrial MnSOD level. a MnSOD is physically associated with Tom20. The subconfluent HEK293 cells as described in Fig. 2a legend were treated with 0.1% ethanol (control) or 10 μM monensin for 24 h in a 10-cm dish before they were collected with a lysis buffer. MnSOD in the total cell supernatant was immunoprecipitated with a plain mouse IgG serving as a control for non-specific binding or a mouse anti MnSOD antibody. The immunoprecipitated MnSOD and Tom20 were identified with rabbit anti MnSOD and Tom20 antibodies through western analysis (n = 3). b, c Knockdown of Tom20 protein by its siRNA significantly reduces monensin-induced increase of mitochondrial MnSOD protein abundance. The cells were typically placed (at a concentration of 1 × 106/10 ml) in a 10-cm dish simultaneously with 60 nM siRNA-Lipofectamine 2000 complex designated for the ethanol control group or at a concentration of 2 × 106/10 ml in a 10-cm dish with 60 nM siRNA-Lipofectamine 2000 complex designated for the monensin group for about 20 h. Then the cells were treated with 0.1% ethanol or 10 μM monensin for 24 h. The mitochondrial (Mito) proteins were separated and analyzed (in b, *p < 0.05 vs the mito EtOH, #p < 0.001 vs the mito monensin group; in c, #p < 0.001 vs the mito EtOH group, *p < 0.05 vs the mito monensin group, n = 6 and two-way ANOVA for both)