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. Author manuscript; available in PMC: 2015 Feb 13.
Published in final edited form as: Cell Rep. 2014 Jan 23;6(3):541–552. doi: 10.1016/j.celrep.2013.12.040

Figure 5. Increased ROS activates TORC2 phosphorylation of Ypk1.

Figure 5

(A) WT (PLY062) and lag1Δlac1Δ + pTet-Lag1 (PLY1501) cells transformed with pPL215 (Ypk1-HA) were grown as in SCD-Ura as in Figure 4H. Cells were harvested and lysed, and the resulting protein extracts were resolved by SDS/PAGE and immunoblotted with α-phospho-Ypk1 (T662), α-HA, and α-G6PDH (as a loading control) antisera. Quantification is relative to untreated WT from each strain, after normalizing to the α-HA signal. (B) Pil1- mCherry tagged W303 co-expressing Slm1-GFP (PLY1555) were grown in SCD minus uracil media, with or without 20 mM NAC pH 8.0 overnight, then treated with DMSO or 1.25 μM Myriocin for 1h. Top section images were collected by confocal microscopy. Inlays depict magnified regions of Slm1 (left), Pil1 (middle), and overlay (right). (C) Colocalization was determined using the Pearson coefficient (ImageJ software).

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