Western Blot Analysis of Protein Expression

The western blot analysis was performed as it is described in [5 (link), 10 (link)]. Briefly, to analyse the expression of proteins in blood, equal amounts of total protein were loaded on a 12% or 4–12% NuPAGE® Novex® Bis–Tris Gel (Invitrogen). To identify the position of specific proteins the Full-Range Rainbow protein molecular weight marker (GE Healthcare Life Science) was loaded on the same gel. After separation by the SDS-PAGE gel proteins were transferred to a Nitrocellulose membrane (Bio-Rad) using a Mini Trans-Blot cell (Bio-Rad). Membranes were incubated with primary antibodies: anti-P-eIF2α (Ser51) (dilution 1:2000) and anti-eIF2α (D7D3) XP (dilution 1:4000), all from Cell Signalling, at 4 °C for overnight. Anti-rabbit secondary antibodies conjugated with horse-peroxidase (Vector Laboratories, Inc.) were diluted to 1:20,000. Signal detection and analysis were performed as it is described in [5 (link), 10 (link)] using Western Lightning ECL Pro development kit (PerkinElmer), HyBlot CL autoradiography film (Denville Scientific) and the ImageQuant TL software (GE Healthcare Life Science). Results are presented as the mean of at least three independent treatment (drug administration) experiments ± SEM.