Fig. 4. SodC is a GPI-anchored membrane protein. (A) A Myc epitope tag was inserted in-frame after the SodC signal peptide. Myc-SodC was expressed in the wild-type background, and detected by western blotting (bottom left). Membrane and cytosolic fractions were prepared, and Myc-SodC was detected only from the membrane fraction (bottom right). (B) Membrane localization of Myc-SodC was also confirmed by indirect immunofluorescence. (C) Log phase wild-type and sodC^– cells were plated at a density of 1x10 cells/cm, and treated with or without 1 unit of GPI-specific PI-PLC for 5 minutes at 25°C. Extracellular SOD activities were compared. (D) The relative levels of extracellular superoxide in wild-type and sodC^– cells were measured using XTT reduction, as described in the Materials and Methods. Virtually identical levels of the radical were detected from three independent experiments. (E) The relative intracellular superoxide levels were determined using NBT. Cell-trapped NBT levels were 18±5% (s.d.) higher in sodC than in wild-type cells (data are from three independent experiments).