Figure 1.

Sub-mitochondrial distribution and PARL-independent maturation of StarD7 in C2C12 cells. (a) Mitochondria and cytosol were separated from C2C12 cells by subcellular fractionation and analyzed by western blotting using anti-StarD7, -SDHA and -GAPDH antibodies. Mt and Cyt indicate mitochondria and cytosol, respectively. (b) Mitochondria isolated from the cells were treated with PBS or alkaline buffer (Na₂CO₃), then the pellet (P) and supernatant (S) were separated by centrifugation. These fractions were analyzed by western blotting with anti-StarD7, porin and CypD antibodies. Porin is a membrane-integrated protein and CypD is a matrix protein. (c) C2C12 cells in proliferation condition were transfected with the expression vector for StarD7 fused with a myc tag at the C-terminus. Cells were permeabilized with 0.005% digitonin (w/v) or 0.1% Triton X-100 (w/v), then immunostained with anti-myc antibody (green). Control means cells without detergent treatment. Mitochondria and nuclei were stained with MitoTracker Red (red) and DAPI (blue), respectively. Bars indicate 5 μm. (d) C2C12 cells were transfected with the expression vector for StarD7 fused with a V5 tag at the C-terminus. After fixation, sections were stained with anti-V5 antibody followed by secondary gold-conjugated antibody. (e) Cell lysates from WT and PARL-KO C2C12 and HEK 293 cells were separated by SDS-PAGE, then the proteins were analyzed by western blotting using anti-StarD7 and PARL antibodies. p, precursor; i, intermediate; m, mature form of StarD7. GAPDH and actin were used as protein loading controls.