Rabbit anti phospho eef2 thr56

Tissue extracts in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1 mM NaVO₃, 5 mM NaF and 1 × protease inhibitor cocktail (Roche, Basel, Switzerland) were analyzed by 4–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes and probed with mouse anti β-tubulin (1:10 000, no. T8328, Sigma-Aldrich, St Louis, MO, USA), rabbit anti-eEF2K (1:500, no. 4661), rabbit anti-phospho-eEF2K (Thr348) (1:500, no. 4411), rabbit anti-phospho-eEF2K (Ser500) (1:500, no. 4451, all from ECM Biosciences, Versailles, KY, USA), rabbit anti-phospho-eEF2 (Thr56) (1:500, no. 2331), rabbit anti-eEF2 (1:500, no. 2332), rabbit anti-phosphor-mTOR (Ser2448) (1:500, no. 5536), mouse anti-mTOR (1:500, no. 4517), rabbit anti-phosphor-p70S6K (Thr389) (1:500, no. 9205) and rabbit anti-p70S6K (1:500, no. 9202) (all from Cell Signaling Technology, Danvers, MA, USA). Immunoreactive bands were developed and quantitated using IRDye secondary antibodies and an Odyssey CLx infrared imager (LI-COR) using conditions recommended by LI-COR (Lincoln, NE, USA).

Tissue extracts in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1 mM NaVO₃, 5 mM NaF and 1X protease inhibitor cocktail (Roche, Basel, Switzerland) were analyzed on 4–12% sodium dodecyl sulfate-polyacrylamide gels, transferred to polyvinylidene difluoride membranes and probed with mouse anti β-tubulin (1:10 000, no. T8328, Sigma-Aldrich, St Louis, MO, USA), rabbit anti-phospho-eEF2 (Thr56) (1:500, no. 2331), and rabbit anti-eEF2 (1:500, no. 2332, Cell Signaling Technology, Danvers, MA, USA). Immunoblots were developed using IRDye secondary antibodies and the protein bands imaged and quantitated using an Odyssey CLx infrared imager (LI-COR, Lincoln, NE, USA).