Anti akt mouse monoclonal antibody

Whole cell lysates were sonicated while on ice to ensure complete disruption of the cellular membranes. Cellular debris was removed via centrifugation at 4,500 × g for 20 minutes at 4 °C. Protein concentration of the supernatants was determined using a BCA assay (Sigma-Aldrich) according to the manufacturer's directions. Multiplex analysis (BioPlex, Bio-Rad) of phosphoproteins involved in insulin signaling in skeletal muscle was carried out using 2 μg protein per replicate. Western blot analysis of Akt phosphorylation (anti-phospho-AKT^ser473 rabbit polyclonal antibody #9271, Cell Signaling, Danvers, MA) as an indicator of insulin signaling was carried out using 60 μg total protein. The C2C12 lysates were resolved by SDS-PAGE and subjected to immunoblotting using infrared detection (Odyssey Imaging System, LI-COR, Lincoln, NE). Total c-JUN or total Akt expression was used as a calibrator (anti-c-JUN rabbit polyclonal antibody #A302-958A, Bethyl Laboratories, Montgomery, TX; anti-Akt mouse monoclonal antibody #2920, Cell Signaling ) in the western blot analyses.