Western Blot was performed using Anti-Phospho-eIF4E (Ser209) Polyclonal Antibody (Product # PA5-17919) and a 30 kDa band corresponding to Eukaryotic translation initiation factor 4E was observed to be either induced in HEK-293 and Hep G2 or increased in HeLa and L6, upon different treatments. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HeLa serum starved (SS) for 16 hours and serum released (SR) with 20% FBS for 30 minutes (Lane 2), HEK-293 (Lane 3), HEK-293 treated with Anisomycin (25 µg/mL for 30 minutes) (Lane 4), L6 (Lane 5), L6 treated with Insulin (200 ng/mL for 10 minutes) (Lane 6), Hep G2 (Lane 7) and Hep G2 treated with Calyculin A (100 nm for 60 minutes) (Lane 8) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The Blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).