Peef2

Mice were decapitated immediately after forced swimming. The frontocortical and hippocampal tissue was collected, and the protein was extracted using standard procedures. Tissue lysates were immediately assessed for the levels of BDNF, eEF₂, CREB and pCREB levels using western blot. After separation on 10% SDS-PAGE gels, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes by electroblotting. BDNF, eEF₂, phosphoeEF₂, CREB, pCREB and β-actin were immunostained by initial incubation with the following primary antibodies: BDNF (1:1000, rabbit polyclonal; Santa Cruz Bio, CA, USA, #sc546), eEF₂ (1:1000, rabbit polyclonal; Cell Signaling Technology; #2332), peEF₂ (1:1000, rabbit polyclonal; Cell Signaling Technology; phospho-eEF₂ (Thr56)#2331), phosphoCREB-ser133 (1:1000, rabbit polyclonal; Cell Signaling, #CST9197S), CREB (1:1000, rabbit monoclonal; Cell Signaling, #CST9191S) and β-actin (1:2000, mouse monoclonal; Transgen Biotech, #HC201). After the membranes were washes using TBST buffer solution, the respective peroxidase-labeled secondary antibodies (anti-rabbit: 1:400; Protein Tech, #SA00001-2; anti-mouse: 1:5000; ZSBG-Bio, #ZB2305) were used for their incubation. Specific band densities were quantified using the Image J software, and the ratio of intensities of pCREB/CREB, peEF₂/eEF₂ and BDNF/β-actin from the same homogenate were calculated^{42 (link)}.

Cells were lysed in Nonidet P-40 (NP-40) buffer (1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1X Complete Mini protease inhibitor cocktail [Roche], 1X phosphatase inhibitor cocktail 2 [Sigma] in PBS). Protein concentrations of lysates were determined using 660 nm Protein Assay Reagent (Thermo Scientific), according to the manufacturer’s protocol. Equal amounts of protein were resolved on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat dried milk in TBS-T (20 mM Tris, 137 mM NaCl, 0.1% Tween-20; pH 7.6) and then incubated in primary antibody. Following primary antibody incubation, membranes were washed three times with 1% non-fat dried milk in TBS-T, incubated in secondary antibody, and then washed three more times with 1% non-fat dried milk in TBS-T. Proteins were visualized by incubating membranes in ECL chemicals and exposing to film. Bands were quantified using ImageJ software. The following primary antibodies were used for western blotting: 4E-BP1, β-tubulin, cyclin D1, eIF2α, eIF4E, eIF4G, GCN2, Ku80, p27, p-4E-BP1, p-eEF2, p-eIF2α, p-p53 (all from Cell Signaling); β-actin (Sigma), and p21 (BD Pharmigen). The secondary antibodies used were anti-rabbit HRP and anti-mouse HRP from Thermo Scientific.