Ng a specific antibody (Niles et al., 2012) that monitors phosphorylation of Ypk1 in the very same internet site (Figure 1–figure supplement 4A). Employing Ypk17A, which also permits facile detection of mobility shifts arising from TORC2-specific phosphorylation (K Leskoske and FM Roelants, unpublished results) (Figure 1–figure supplement 4B), we followed the kinetics of this alter. Loss of TORC2-mediated Ypk1 phosphorylation upon hyperosmotic shock occurs very quickly (inside 1 min) and persists for about 15 min (Figure 1D), but is transient. By 20 min right after hyperosmotic shock, TORC2-mediated Ypk1 phosphorylation is once again detectable and is nearly back for the pre-stress level by 75 min (Figure 1–figure supplement 5A). Speedy reduction in TORC2-mediated Ypk1 phosphorylation below hypertonic strain was nevertheless observed in mutants lacking the Sho1- or Sln1-dependent pathways that converge on Hog1 or HogMuir et al. eLife 2015;4:e09336. DOI: 10.7554/eLife.2 ofResearch advanceBiochemistry | Cell biologyFigure 1. Fps1 (but not Gpt2) is phosphorylated by Ypk1. (A) Wild-type (BY4741) or ypk1-as ypk2 (Eprazinone Purity & Documentation yAM135-A) cells expressing plasmid borne Gpt2-3xFLAG (pAX238) or Gpt23A-3xFLAG (pAX244) had been grown to mid-exponential phase and after that treated with car (-) or ten M 3-MB-PP1 (+) for 90 min. Cells have been 475207-59-1 supplier harvested, extracts prepared, resolved by SDS-PAGE, and blotted as in `Materials and methods’. (B) Wild-type cells expressing either Fps1-3xFLAG (yGT21) or Fps13A-3xFLAG (yGT22) from the FPS1 promoter at the standard chromosomal locus, or ypk1-as ypk2 cells expressing either Fps1-3xFLAG (yAM281) or Fps13A-3xFLAG (yAM284-A) in the FPS1 promoter at the regular chromosomal locus, were grown to mid-exponential phase and treated as in (A) with vehicle or 3-MB-PP1 for 60 min. Cells had been harvested, extracts ready, resolved by Phos-tag SDS-PAGE, and blotted as in `Materials and methods’. Unphosphorylated Fps1 (red asterisk). (C) A tor2-as strain (yKL5) expressing Fps1-3xFLAG (pAX274) or Fps13A-3xFLAG (pAX275) was grown to mid-exponential phase after which treated with vehicle (-) or 2 M BEZ-235 (+) for 30 min. Cells were harvested, extracts prepared, resolved and analyzed as in (B). (D) Wild-type (BY4741) or tor2-29ts (JTY5468) cells expressing Ypk17A-myc (pFR252) were grown at 30 (left panel) or 26 (suitable panel) to mid-exponential phase, then diluted into fresh YPD inside the absence (-) or presence of 1 M sorbitol (final concentration). Immediately after the indicated occasions (15 min), culture samples have been collected, lysed along with the resulting extracts resolved by Phos-tag SDS-PAGE and analyzed by immunoblotting with anti-myc mAb 9E10, as described in `Materials and methods’. (E) As in (D), except for the genotype (strain) expressing Ypk17A-myc (pFR252), which have been, aside from the wild-type control, hog1 (YJP544), sho1 (JTY5540), ssk1 (JTY5541), ssk22 (JTY5539), ssk2 (JTY5538) or pbs2 (JTY5537), plus the therapy with 1 M sorbitol was for 1 min. (F) Wild-type (BY4741) or otherwise isogenic cna1 cna2 (JTY5574) cells expressing Ypk17A-myc (pFR252) have been grown to mid-exponential phase then diluted into fresh YPD within the absence (-) or presence (+) of 1 M sorbitol (final concentration). Following 1 min, the cells were collected, lysed plus the resulting extracts resolved by Phos-tag SDS-PAGE and analyzed by immunoblotting with anti-myc mAb 9E10, as described in `Materials and methods’. (G) Wild-type cells expressing either Fps1-3xFLAG (yGT21) or Fps13A-3xFLAG (yGT22) from the chromosomal FPS1 locus, were.