Rabbit α eif4e

Protein samples for the cap-affinity assay were separated by 8-15% gradient SDS-PAGE (polyacrylamide gel electrophoresis). Ten or twelve percent SDS-PAGE was used for PARP and eIF4E proteins, respectively. Following protein transfer to PVDF (Amersham Biosciences) the membranes were blocked in 5% non-fat dry milk for 1 hour at room temperature in Tris-buffered saline-Tween (TBST: 0.15 M NaCl; 0.01 M Tris-HCl, pH 7.6; 0.05% Tween 20). The membranes were then incubated for 1 hour at ambient temperature or overnight at 4°C with the chosen primary antibody. The primary antibodies employed were rabbit α-eIF4E (catalog number 9742), rabbit α-PARP (catalog number 9542), α-SV40 large T antigen (catalog number 15729) all from Cell Signaling Technology (Danvers, MA) at a 1:1000 dilution, mouse α-β-actin (Sigma-Aldrich, catalog number A1978) at a 1:10,000 dilution and rabbit α-eIF4GI (kindly provided by Nahum Sonenberg, McGill University Montreal, QC, Canada) at a 1:2500 dilution. Preceding and following incubation with the appropriate horseradish peroxidase labeled secondary antibodies, the blots were washed three times for 5 minutes in TBST and detection was then performed utilizing ECL Plus Western Blotting System (Amersham Biosciences) to visualize the bands of interest.