Torin 1

Cells were washed once with ice-cold wash buffer (40 mM HEPES [pH 7.4], 150 mM NaCl), and lysed in ice-cold CHAPS buffer (40 mM HEPES [pH 7.4], 150 mM NaCl, 2 mM EDTA, 0.3% CHAPS, phosphatase inhibitor cocktail and protease inhibitor cocktail). The cell debris was removed by centrifugation at 13,000 rpm for 10 min in a microfuge. The soluble fractions of cell lysates were mixed with anti-raptor antibody (4 μg/10 cm dish, Life Technology, Cat. 42–4000, RRID: AB_2533523), and the mixtures were incubated with rotation for 1.5 hr at 4°C. 80 μl of a 50% slurry of protein A/G plus-sepharose (Santa Cruz Biotechnology) was then added and the incubation continued for an additional 1 hr. Immunoprecipitates were washed twice with ice-cold CHAPS buffer, and once with mTOR kinase buffer (25 mM HEPES [pH 7.4], 50 mM KCl, 10 mM MgCl₂). The kinase assays were performed as previously described (Kim et al., 2002 (link)). CaM (2 μM) or/and CaCl₂ (1 mM) were added into the kinase reaction as indicated. CMDZ (8 μM) or Torin 1 (100 nM, Cayman Chemical) was incubated with the reaction mixtures 10 min prior to initiating the reaction by addition of 250 μM ATP (Sigma). The phosphorylation states of S6K or 4EBP1 were detected by immunoblotting.

Antibodies used for immunoblotting included anti-sestrin2 obtained from Proteintech Group, anti-phospho-p70 S6 Kinase, anti-phospho-S6 Ribosomal Protein, anti-S6 Ribosomal Protein, anti-E-cadherin, anti-N-cadherin, and anti-TSC2 from Cell Signaling Technology, anti-p70 S6 Kinase from Santa Cruz Biotechnology, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Aviva Systems Biology, and anti-β-Actin from Developmental Studies Hybridoma Bank. Rapamycin was obtained from LC Laboratories. Torin 1 was purchased from Cayman Chemical. N-acetyl cysteine (NAC) was purchased from Sigma-Aldrich.

Pharmacological inhibitors were stored in DMSO (Fisher Scientific) stocks. Embryos were treated with drugs, 0.5 µM or 1 µM rapamycin (Santa Cruz Biotech) or 0.5 µM Torin 1 (Cayman Chemical), diluted in egg water (1.5 mL stock salts added to 1 L distilled water) and supplemented with 0.003% 1-phenyl 2-thiourea (PTU) (Sigma) and DMSO to 1% final. Control embryos were incubated with 1% DMSO vehicle. Embryos were dechorionated and treated with DMSO, rapamycin or Torin 1 at 2.5 dpf until 5 dpf and 7dpf.

GC7 (Merck Millipore) was added for 24 hours to B cells (10 μM) on day 2 after B cell stimulation (unless otherwise indicated) or to Jurkat/NIH 3T3 cells (100 μM). 10 nM bafilomycin A1 (Cayman Chemical), 10 μg/ml cycloheximide (Sigma), or 100 nM Torin 1 (Cayman Chemical) were added to cells for 2 hours. 10 μM etoposide (Cayman Chemical) or 1 μM thapsigargin (Cayman Chemical) was added to cell culture for 6 hours, or 1 mM difluoromethylornithine (DFMO, Enzo Life Sciences) for 24 hours.