Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HEK-293 (Lane 1) and HEK-293 treated with Insulin (100 nM, for 10 min) (Lane 2). The blots were probed either, with Anti-Phospho-Phospho-AS160 (Ser558) Recombinant Rabbit Monoclonal Antibody (Product # 702305, 1 µg/mL). To confirm the specificity competition was performed with the phospopeptide (10 µg/mL) (Lane 3 & 4) and non phospopeptide (10 µg/mL) (Lane 5 & 6). A 160 kDa band corresponding to Phospho-AS160 (Ser558) was observed across the cell lines tested, which specifically gets competed with phosphopeptide and not with the nonphosphorylated peptide. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 dilution). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with wet transfer method. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).