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. 2015 Feb 2;208(3):299–311. doi: 10.1083/jcb.201410076

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© 2015 Roelants et al.

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Figure 3. — Phosphorylation by Gin4 down-regulates Fpk1 activity. (A) Cells expressing either wild-type Fpk1 (BY4741, WT) or Fpk1^11A (YJW2) and also expressing Ypk1-myc from the GAL1 promoter (pAM76) were grown to mid-exponential phase, untreated or treated with Myr (1.25 μM), and lysed. The resulting extracts were resolved by SDS-PAGE and analyzed by immunoblotting with anti–c–myc mAb 9E10. (B) Cells expressing wild-type Fpk1 (BY4741, WT), an isogenic strain lacking both Fpk1 and Fpk2 (YFR205, fpk1Δ fpk2Δ) or expressing Fpk1^11A (YJW2) and also expressing Dnf1(1403–1571)–myc from the GAL1 promoter (pES10) were grown to mid–exponential phase and lysed. The resulting extracts were resolved on a Phos-tag gel and analyzed by immunoblotting with anti–c–myc mAb 9E10. (C) Wild-type (BY4742, WT), fpk1Δ (JTY6581), Fpk1^11A (YFR307), Fpk1^11A dnf1Δ (YFR308), or Fpk1^11A dnf1Δ dnf2Δ (YFR312) strains, as indicated, were plated as a lawn on YPD plates and then overlaid immediately at the center with a sterile filter paper disk onto which a solution containing the indicated amounts of Myr had been spotted. After incubation at 30°C for 2 d, plates were photographed.