Blotto solution

Samples were sonicated at 40 kHz for 20 s, heated at 99 °C for 5 min and centrifuged at 14,000 rpm for 5 min at room temperature; 20 μg of protein (from insulin response assays samples) or 10 μg of protein (from Akt CoIP samples for mass spectrometry validation), were electrophoresed on SDS-PAGE (10–12% gels) and separated proteins were electro-transferred to PVDF membranes. Blots were blocked with 5% Blotto solution (sc-2325, Santa Cruz) in TBST for 1 hour, and incubated overnight at 4 °C with primary antibodies. Blots were washed 3 times and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Antibody binding was visualized with ECL (Enhanced Chemiluminescence) reagent (Immobilon^™ Millipore Corporation) and quantified with ImageJ software (National Institutes of Health). Primary antibodies: Akt 1/2/3 (sc-8312, 1:5000), pAkt Ser473 (sc-7985, 1:3000), 14-3-3 ζ (sc-1019, 1:5000) and Hsp60 (sc-13115, 1:1000), were purchased from Santa Cruz Biotechnology; pAkt Thr308 (9275S, 1:3000) from Cell Signaling; GAPDH (AM4300, 1:35000), from Thermo Fisher Scientific. Actin (mouse monoclonal, 1:8000) was provided by Dr. J.M. Hernandez from Cell Biology Department, CINVESTAV, Mexico. Secondary antibodies: HRP-goat anti-mouse IgG (115-035-003, 1:10000) and HRP-goat anti-rabbit IgG (111-035-144, 1:10000) were purchased from Jackson ImmunoResearch.