FIGURE 5.

Alkaline carbonate extraction of CHO cell mitochondria expressing epitope-tagged rat MTHFD2L. CHO cell mitochondria were prepared and subjected to Na₂CO₃ or NaCl or Triton X-100 extraction as described under “Experimental Procedures.” The insoluble integral membrane proteins (pellet; P) were separated from the soluble and peripheral membrane proteins (supernatant; S) by ultracentrifugation. Equal concentrations of pellet and supernatant proteins were fractionated on a 10% SDS-polyacrylamide gel and subjected to immunoblotting with anti-V5 (1:4000 dilution), anti-manganese superoxide dismutase (MnSOD) (peripheral membrane protein marker; 1:2000 dilution), or anti-porin (integral membrane protein marker; 1:2000 dilution) antibodies. MTHFD2L migrated at an apparent molecular mass of 37 kDa, manganese superoxide dismutase migrated at an apparent molecular mass of 25 kDa, and porin migrated at an apparent molecular mass of 31 kDa.