Fig. 2.
Requirements for modification of IMPDH in vitro. Extract from DY1107 (TAP-IMD3), DY424 (TAP-IMD2), or DY2040 (TAP-IMD2C335A) was incubated at 30°C with the indicated substrates ± 47 μM MPA (A) or varying amounts of MPA in the presence of IMP and NAD+ (B). (C) Strain DY424 was incubated in synthetic complete (SC) media containing 15 μg/ml MPA for 120, 150, or 240 min as indicated. An aliquot of cells treated for 120 min was washed twice with half vol of water for 20 min (120 min + 20 min wash), and returned to 30°C in SC media lacking drug for 30 or 120 additional min (120 min + 50 min wash; and 120 min + 140 min wash) and analyzed by Western blotting vs. the TAP-tag.