TCA precipitation protocol was followed for total protein extracts (Caspari et al., 2000 (link)). The following dilutions of antibodies were used in this study: 1:100 anti-Tor1, 1:100 anti–P-Tor1.T1972, 1:100 anti–P-Tor2.S1975, 1:1,000 anti–P-Gad8.S546, 1:100 anti–P-Gad8.T387, 1:100 anti-Gad8 antibodies, 1:2,000 Phospho-(Ser/Thr) Akt substrate (PAS) antibody (Cell Signaling Technology), 1:100 p70 S6Kα (Santa Cruz Biotechnology, Inc.), 1:100 P-S6Kα.T389 (Cell Signaling Technology), and 1:1,000 S6 antibody (Abcam). 1:100 mTOR antibodies were from EMD Millipore. 1:100 AKT and 1:100 P-AKT.S473 antibodies were from Cell Signaling Technology. Anti–p-Tor1.T1972, anti–P-Gad8.S546, and anti–P-Gad8.T378 were all generated by Eurogentec. Anti–P-Tor2.S1975 antibodies were used to detect both S. pombe Tor2 and mTOR phosphorylation and were generated by Eurogentec. Anti-Tor1 antibodies were raised against the unique Tor1 sequence (aa 2,231–2,274). Alkaline phosphatase– or peroxidase (only for loading control in Figs. 1 D , 3 A , and 5 I )-coupled secondary antibodies were used for all blots followed by direct detection with NBT/BCIP (VWR International) substrates on polyvinylidene fluoride membranes.
Protocol detail
TCA Precipitation and Antibody Dilutions
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Alkaline phosphatase
Anti antibodies
Anti p antibodies
Antibodies
Antibody
Dilutions
Figs
Membranes
Mtor
Peroxidase
Phosphorylation
Polyvinylidene fluoride
Protein
S pombe
Corresponding Organization :
Other organizations : University of Manchester, Cancer Research UK Manchester Institute
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Variable analysis
independent variables
- Antibody dilutions used in the study:
- 1:100 anti-Tor1
- 1:100 anti–P-Tor1.T1972
- 1:100 anti–P-Tor2.S1975
- 1:1,000 anti–P-Gad8.S546
- 1:100 anti–P-Gad8.T387
- 1:100 anti-Gad8 antibodies
- 1:2,000 Phospho-(Ser/Thr) Akt substrate (PAS) antibody (Cell Signaling Technology)
- 1:100 p70 S6Kα (Santa Cruz Biotechnology, Inc.)
- 1:100 P-S6Kα.T389 (Cell Signaling Technology)
- 1:1,000 S6 antibody (Abcam)
- 1:100 mTOR antibodies (EMD Millipore)
- 1:100 AKT and 1:100 P-AKT.S473 antibodies (Cell Signaling Technology)
- Protein expression and phosphorylation levels measured using the specified antibodies
- TCA precipitation protocol was followed for total protein extracts (Caspari et al., 2000)
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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