2.6. Western Blot

Semi-dry immunoblotting was performed as described previously [24]. Briefly, 30 µg of total protein lysate was separated using SDS polyacrylamide gel electrophoresis and proteins were blotted on Immobilon-P PVDF membranes (0.45 µm; Millipore, Burlington, MA, USA). For the assessment of whether the protein transfer was successful, membranes were reversibly stained using Ponceau S (Sigma Aldrich, St. Louis, MO, USA). Primary antibodies (anti-eIF4E-antibody product# 9742; anti-eIF4AI-antibody product# 2490; anti-GAPDH-antibody product# 2118; all three Cell Signaling Technology; anti-Actin-antibody product# A2103, Sigma Aldrich) were diluted 1:1000 in Tris-buffered saline supplemented with 0.1% Tween (TBS-T) containing 5% bovine serum albumin (BSA fraction V, Roche Diagnostics) and incubated overnight at 4 °C. Subsequently, horseradish peroxidase conjugated secondary anti-rabbit antibody (1:5000 dilution in 5% non-fat dried milk in TBS-T; ECL™ Anti-rabbit IgG HRP; GE Healthcare, Chicago, IL, USA) was incubated for 1 h at room temperature. For puromycin detection, anti-puromycin antibody (clone 12D10, product# MABE343; MERCK KGaA, Darmstadt, Germany) was diluted 1:5000 in TBS-T containing 5% bovine serum albumin and incubated overnight at 4 °C. Subsequently, horseradish peroxidase-conjugated secondary anti-mouse antibody (1:3000 dilution in 5% non-fat dried milk in TBS-T; ECL™ Anti-mouse IgG HRP; GE Healthcare) was incubated for 1 h at room temperature.

The visualization of proteins of interest via chemiluminescence using Amersham ECL Select Western blotting detection Reagent (GE Healthcare) was performed using the ImageQuant^TM LAS 500 (GE Healthcare). The ImageJ software [25] was used for densitometrical analysis and the signals were normalized to GAPDH and Actin as a loading control.