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. 2009 Sep 24;18(23):4552–4564. doi: 10.1093/hmg/ddp421

Figure 2.

Figure 2.

Expression and localization of IMS-targeted hSOD1 in NSC34 cells. (A) Western blot of cellular homogenates from cells stably expressing WT and G93A IMS-hSOD1 using a polyclonal anti-SOD1 antibody that recognizes both the human and the mouse protein. mSOD1, mouse SOD1; P1 and P2, precursor products of incomplete protein cleavage; M, mature, fully processed hSOD1. (B) Western blot of enriched mitochondrial fractions from NSC34 cells stably transfected with WT or mutant IMS-hSOD1. Each lane contains 50 µg of protein. The mitochondrial outer membrane protein VDAC1 is used as a loading control. Note that mature G85R mutant IMS-hSOD1 is masked by endogenous mSOD1. (C) Western blot of mitochondria from cells stably expressing G85R IMS-hSOD1 using a human specific anti-SOD1 antibody (first lane). Proteinase K (PK) treatment does not completely digest mutant hSOD1 (second lane), but digestion is almost complete when mitochondrial membranes are solubilized with detergents (TX, third lane). (D) Western blot of mitochondria form G85R IMS-hSOD1 cells after hypo-osmotic swelling. PK treatment fully digested mutant hSOD1 only when the outer mitochondrial membrane was disrupted. (E) SOD1 native activity gel assay of enriched mitochondrial fractions from cells stably expressing IMS-hSOD1. hSOD1, purified human SOD1; mock, cells transfected with empty plasmid. (F) Western blot of supernatant (SN) and pellet (P) fractions of cells expressing WT untargeted or IMS-hSOD1 after digitonin (DG) treatment. A human specific anti-SOD1 antibody demonstrates the presence of hSOD1 only in the pellet fraction of cells expressing IMS-hSOD1. Longer exposure of the same blot shows small amounts of hSOD1 in the supernatant after DG treatment when IMS-hSOD1 is expressed. Cytochrome c and Akt antibodies are used as controls for IMS and soluble cytosolic protein fractions, respectively.