The positive transformants were screened on MD medium and analyzed by PCR.
(D) Atg31 protein was isolated by GST-tag purification from yeast grown in full or SD-N medium and analyzed by mass spectrometry.
Coverslips have been mounted in Dako mounting medium and analyzed with a Leica confocal microscope.
Embryos were washed in embryo medium and analyzed under a fluorescence microscope.
Cells on coverslips were mounted on slides with Dako mounting medium and analyzed with Zeiss LSM510 and Leica SP2 AOBS confocal microscope.
Coverslips were mounted on microscope glass slide with DAKO mounting medium and analyzed using immunofluorescent confocal microscopy or laser scanning cytometry (CompuCyte Corp), essentially as previously described [28].
Equal number of live cells were suspended at 3 5×106 cells/ml in complete DMEM medium and analyzed at 37°C using a Clarke electrode.
Microfossil slides were prepared using permanent mounting medium and analyzed using a Nikon microscope with phase-contrast illumination at 1000× magnification [29], [30].
Cells were stained with 0.5 µM JC-1 and kept at 37°C for 20 min, washed and resuspended in a total volume of 500 µL complete medium and analyzed by Cyan ADP (Coulter, Brea, CA).
Clones expressing the neo-resistant gene were selected by including G418 (350 µg/ml; Invitrogen, Carlsbad, CA) in the cell culture medium and analyzed for target gene homologous recombination by Southern blot analysis of genomic DNA.
