Phospho eef2 thr56

Cells were seeded at a density of 3x10⁵ cells in a 10 cm dish in complete media overnight followed by treatment with vehicle or drug for the indicated times. Cell lysates were prepared using a NP-40 lysis buffer (include recipe) including protease and phosphatase inhibitors (Roche). Protein concentrations were determined and equal amounts for each sample were analyzed by SDS-PAGE. Primary antibodies used for immunoblot analysis were: FKBP12 (#3635–100, Biovision), eIF2 alpha (#9722, Cell Signaling Technologies), phospho-eIF2 alpha (Ser51) (#3398, Cell Signaling Technologies), eEF2 (#2332, Cell Signaling Technologies), phospho-eEF2 (Thr56) (#2331, Cell Signaling Technologies), Akt (#9272, Cell Signaling Technologies), phospho-Akt (Ser473) (#9271, Cell Signaling Technologies), phospho-Akt (Thr450) (#9267, Cell Signaling Technologies), mTOR (#2983, Cell Signaling Technologies), RACK1 (#610178, BD Biosciences), p70 S6 kinase (#2708, Cell Signaling Technologies), 4E-BP1 (#9644, Cell Signaling Technologies), phospho-4E-BP1 (Ser 65) (#9451, Cell Signaling Technologies).

Primary cortical cultures were fixed in cold 4% formaldehyde solution in phosphate saline buffer 0.01M (PBS) for 10 min. The cells were washed three times for 5 min with PBS+ Triton X-100 1%. The cells were then incubated for 1 h at room temperature (RT) in blocking solution of PBS + Triton X-100 1%, containing 10% fetal calf serum and 0.3% bovine serum albumin (BSA). The cells were incubated overnight at 4°C and 1 h at room temperature (RT) with the following primary antibodies diluted in the same blocking solution: phospho-eEF2 Thr56 (1:100, Cell Signaling), eEF2 (1:100, Cell Signaling), MAP2 (1:1000 Abcam), puromycin (1:1000, Millipore). Cells incubated in blocking solution lacking the primary antibody were used as negative control. After washing with PBS+1% Triton (3 × 5 min), cells were incubated for 1 h at RT with the corresponding secondary antibodies: donkey Anti-Chicken AlexaFluor^® 488 (1:500), donkey Anti-Rabbit and Anti-Mouse AlexaFluor^® 594 (1:500). The cells were then washed with PBS+1% Triton (2 × 5 min) and PBS (2 × 5 min). Finally, coverslips were mounted on Superfrost^TM Plus Adhesion slides (Thermo Fisher Scientific) with Slow Fade^® Gold antifade reagent containing DAPI (Life Technologies).