P eif4g

Cortical neurons and brain tissue were homogenized in lysis buffer containing NaCl 137 mM, KCl, 2.7 mM, Na₂HPO4 10 mM, KH₂PO₄, 1.8 mM, EDTA 5 mM, Triton 1%, and complete protease and phosphatase inhibitors (Roche Applied Science). Immunoblotting was done with HRP-conjugated secondary antibodies and Pierce ECL Western Blotting Substrate. The following primary antibody was used in this study: p-eIF4E (Abcam, ab76256 1:1000), eIF4E (Abcam, ab47482 1:1000), p-ERK1/2 (Cell Signaling, 4370S 1:1000), ERK1/2 (Cell Signaling, 4695S 1:1000), p-eIF4G (Cell Signaling, 2441S 1:1000), eIF4G (Cell Signaling, 2498 1:1000), p-MNK1 (Cell Signaling, 2111, 1:1000), MNK1 (Cell Signaling, 2195S, 1:1000), GAPDH (Cell Signaling, 5174 1:2000) and calnexin (Stressgen, SPA-865 1:2000). Loading controls were run on the same gel, and for some experiments Mini PROTEAN® TGX Stain-Free™ Gels (Bio-Rad) were used as loading controls. Signals were acquired using an image analyzer (Bio-Rad, ChemiDoc MP Imaging System) and images were analyzed and prepared using ImageJ.
For additional measurements of eIF4E phosphorylation state, the AlphaLISA SureFire Ultra p-eIF4E (Ser209) Assay Kits (PerkinElmer) were used according to the manufactures protocol. AlphaLISA signals were measured using a Tecan SPARK plate reader on recommended settings.