Western Blot was performed using Anti-Phospho-PRAS40 (Thr246) Polyclonal Antibody (Product # PA5-17175) and a 40 kDa band corresponding to Proline-rich AKT1 substrate 1 was observed across cell lines tested. Whole cell extracts (30 µg lysate) of HEK-293 (Lane 1), HEK-293 treated with serum starvation (14Hrs) followed by insulin treatment (15 nm,20 min) (Lane 2), HEK-293 treated with serum starvation (14Hrs) followed by rapamycin treatment (10 nm,30 min) (Lane 3), HeLa (Lane 4), HeLa treated with serum starvation (14Hrs) followed by insulin treatment (15 nm,20 min) (Lane 5), HeLa treated with serum starvation (14Hrs) followed by rapamycin treatment (10 nm,30 min) (Lane 6), NIH/3T3 (Lane 7), NIH/3T3 treated with serum starvation (14Hrs) followed by insulin treatment (15 nm,20 min) (Lane 8) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). 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).