FIG. 2.

All isoforms of thioredoxin are extracted with ethanol. Log-phase cells (30 A₆₁₀ units) of S. cerevisiae CY4 carrying either TRX1-3HA, TRX2-3HA, or TRX3-3Myc were suspended in 1 ml of 20% ethanol at 37°C for 2 h. The amounts of thioredoxin in the resultant supernatants were determined by Western blotting using anti-HA antibody 6E2 (α-HA) (Cell Signaling Technology) (left panel) or anti-Myc antibody A-14 (α-Myc) (Santa Cruz Biotechnology) (right panel). Lanes M, molecular weight markers; lanes 1, CY4 without tagged thioredoxin; lanes 2, CY4 with Trx1-3HA; lanes 3, CY4 with Trx2-3HA; lanes 4, CY4 with Trx3-3Myc. Since the thioredoxins were tagged with three copies of each epitope, they are slightly larger than the originals (11 to ∼14 kDa).