Imagequant

For northern blot analysis, log phase YPD grown cells were washed and transferred to YNB medium containing 10 mM of the different D-amino acids or ammonium sulfate. RNA was extracted from yeast cells using Trizol (Invitrogen, Carlsbad, CA). ³²P-labelled DNA probes for northern blots were synthesized from PCR products generated using gene specific primer pairs (S2 Table). The probe for each DAO gene did not cross hybridize amongst each other. For quantitative northern analysis, blots were hybridized with the indicated probe, stripped and hybridized with an ACTIN probe. The blot was exposed to Phosphorimager Screen and quantified with ImageQuant (Molecular Dynamics). Signal of each DAO gene was normalized to that of the ACTIN gene and expressed as the relative amount to H99 or R265. For quantitative RT-PCR, log phase YPD grown cells were washed, transferred to YNB medium and grown for 3 h. Cells were washed and transferred to YNB medium containing 10 mM of the different D-amino acids or ammonium sulfate for 2 h. RNA isolation and quantitative RT-PCR was performed as described [26 (link)]. The PCR efficiency and CT determination was performed using the algorithm as described [30 (link)]. Data were normalized with ACTIN1 levels and expressed as the amount relative to RNA levels of H99 or R265 grown in ammonium sulfate.

Whole‐cell extracts from HA‐treated HPF and HGF cells were prepared by lysis in RIPA buffer as described.30 Lysates were run on 10% SDS‐PAGE, and transferred to Amersham^™ Protran^® membrane (Sigma, Basel, Switzerland). Proteins of interest were visualized using anti‐phospho‐Akt, anti‐Akt, anti‐phospho‐Erk1/2, anti‐Erk, anti‐phospho‐p38, anti‐p38 (all from Cell Signaling Technology, Danvers, MA, USA), and anti‐vinculin (Sigma) antibodies followed by horseradish peroxidase‐conjugated secondary antibodies (MP Biomedicals, Santa Ana, CA, USA) for detection with the SuperSignal^™ West Dura Substrate (ThermoFisher Scientific, Zug, Switzerland). Phospho‐Akt, phospho‐Erk1/2 or phospho‐p38 protein expression relative to the respective total protein control was quantified by densitometry using ImageQuant (Molecular Dynamics, Groningen, The Netherlands). Data represent means ± SD from three independent experiments performed with three different cell donors.

SDS electrophoresis of the muscle extracts was performed as described (Danieli Betto et al., 1986 (link)) in a discontinuous buffer system with a 4%T stacking gel, pH 6.8 and a 6%T, constant concentration, 37% w/v glycerol, pH 8.8, running gel, respectively. Samples (4 μg) were separated at 100 V, overnight. Gels were stained with SYPRO Orange (Molecular probes) and scanned at 570 nm with a Typhoon laser densitometer (GE Healthcare). Quantitation was achieved using ImageQuant (Molecular Dynamics) software. MyHC-specific isoforms were identified by electrospray ionization/tandem mass spectrometry (ESI-MS/MS), as described previously (Moriggi et al., 2010 (link)).