from PMID 17560372 ==Materials==* NTCB dissolve to 7.5 mM in water (* '''Urea Lysis Buffer''': 50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi* '''PPi''': 10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease inhibitor cocktail and 1 mM PMSF.
Kinase Assay==Protocol==# Grow cells to mid log phasePPi: 10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease # Dilute to OD = 0.2 in YPD with or without inhibitor cocktail and 1 mM PMSFtreatments.# Incubate 1h at 24C in YPDCultures were mixed with # Add TCA (to a final concentration of 6%(180 uL for 3 mL culture volume) and put # Place cells on ice for 5 min# Spin cells at least 5000 RPM for 5 min before cells were pelleted.# Wash with 1 mL Acetone, washed twice spin and wash with cold acetone, again and dried aspirate acetone.# Dry cells in a speed-vac. Cell lysis was done in 100 μl of urea buffer (50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi) with glass beads in a bead beater with subsequent heating for 10 30 min to 65°C. For NTCB cleavage 30 μl of 0.5 M CHES (pH 10.5) and 20 μl of NTCB (7.5 mM # Resuspend in H2O) were added and samples incubated over night at RT before 1 vol 150 uL of 2× sample buffer (+20 mM TCEP and 0.5× PPi) was added. Further analysis was done by SDS-PAGE and immunoblotting using anti-HA antibody 12CA5 or anti-T570-P antiserumUrea Lysis Buffer. from PMID 19748353 Sch9 Phosphorylation Analyses To analyze Sch9T570A-HA5 C-terminal phosphorylation, we used the chemical fragmentation analysis as described previously ([Urban et al., 2007] and [Wanke et al., 2008]). For quantifications of Sch9 phosphorylation, NTCB-cleaved extracts were separated by 7.5% SDS-PAGE followed by immunoblotting # Lyse with anti-HA antibody 12CA5. The anti-HA antibody was detected with far-red fluorescent Alexa Fluor 680 dye-labeled secondary anti-mouse antibody a beadbeater (Invitrogen, A21057), and fluorescence intensity was measured using the Odyssey Infrared Imaging System (LI-COR3x 30s). from PMID 18513215 Sch9 and Ypk2 carboxy-terminal phosphorylation To analyse Sch9-5HA C-terminal phosphorylation, TB50 cells containing plasmids pJU450 and pJU676 were grown in SC-Ura, -His, -Leu to mid-log phase, harvested and re-suspended in YPAD + 0.2% Gln # Heat at 0.5 OD600. Cells were grown 65C for 60 min at 30°C prior to addition of medium containing rapamycin or caffeine and subsequent incubation for another 30 10 min. Chemical fragmentation analysis was done as described (Urban et al., 2007). To analyse Ypk2 phosphorylation, MP8 cells were grown in YPD + 0.# Spin 2% glutamine min at 30°C to an OD600 between 0high in eppendorf centrifuge.6 # Remove 100 uL and add 30 uL of 0.8, at which point rapamycin or caffeine was added to the indicated final concentration5M CHES and 20 uL of NTCB. Cells were shaken for Save an additional 30 min and then harvested untreated lysate sample as described in Urban et alwell. (2007), but without 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage# Incubate overnight at room temperature. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose membrane # Add lysis buffer and immunoblotted with anti-blot using HA antibody or rabbit anti-phospho-T659 Ypk2 antiserum (this antiserum detects both Sch9 phosphorylated at T737 by TORC1 as well as Ypk2 phosphorylated at T659 by TORC2; R. Loewith, unpublished)antibodies.
