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. 2014 Aug 13;111(34):E3544–E3552. doi: 10.1073/pnas.1413201111

Fig. 4.

Fig. 4.

Calcineurin can activate protective and toxic substrates in yeast. (A) The HiTox yeast strain was spotted onto plates containing uninducing media (SD −Leu; vector, hph1, slm2, or crz1 selective; Lower) and replica plated in threefold serial dilutions onto α-syn–inducing plates containing selective media and SGal −Leu (Upper). (B) Peak intensity of phosphopeptide ENVD(phospho)SPR from Slm2p relative to control using SRM-mass spectrometry after phosphopeptide enrichment as described (66). Error bars reflect biological and technical variability. *P < 0.05 (Student t test). (C) Similar spotting assays as described in A but onto plates containing uninducing media [SD −Leu; slm2, slm2-AEFYAE (unable to bind calcineurin) selective; Lower] and replica plated in threefold serial dilutions onto α-syn–inducing plates containing selective media and SGal −Leu (Upper). (D) Real-time PCR for the TORC2-dependent genes ylr194c and dia1 in control and HiTox yeast strains in the presence of slm2 or a low dose of FK506 (25 μg/mL). (E) Peak intensity of phosphopeptide MDSANS(phospho)SEKISK from Crz1p relative to control using SRM-mass spectrometry after phosphopeptide enrichment as described (66). Error bars reflect technical variability. *P < 0.05 (Student t test). (F) The HiTox yeast strain was spotted onto plates containing uninducing media [SD −Leu; vector, crz1, PVIVIT-crz1 (high affinity to calcineurin) selective; Lower] and replica plated in threefold serial dilutions onto α-syn–inducing plates containing selective media and SGal −Leu (Upper) in the presence and absence of crz1 (crz1Δ). (G) β-Glactosidase luminescent assay for control and HiTox strains harboring a reporter for crz1p-dependent transcriptional activity in the presence and absence of 25 μg/mL FK506, calcineurin (cnb1Δ), and/or BSCaMIQ.