Goat anti mouse or goat anti rabbit alexa fluor 680

Protein extracts were prepared by lysing cells cultured in six‐well plates with radioimmunoprecipitation assay (RIPA) buffer (50mM Tris, 150mM NaCl, 1mM ethylenediamine tetraacetic acid [EDTA], 0.5% NaDOAc, 0.1% sodium dodecylsulfate [SDS], and 1.0% NP‐40) plus a phosphatase and protease inhibitor cocktail (Xpert P3200‐001 and P3100‐001; GenDEPOT, Katy, TX, USA). Protein concentration was determined by the Bio‐Rad method (DC Protein Assay), and equal amounts of proteins were subjected to SDS–polyacrylamide gel electrophoresis (SDS‐PAGE) gels (8–15%). Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and incubated overnight with primary antibody at 4°C, followed by a 1‐h incubation with secondary goat anti‐mouse or goat anti‐rabbit Alexa‐Fluor 680 (Molecular Probes, Eugene, OR, USA), or donkey anti‐goat 780 (LI‐COR Biosciences, Lincoln, NE, USA) antibody. Primary antibodies include total protein kinase B (Akt), phospho‐Akt (Ser473) (Cell Signaling Technology, Beverly, MA, USA) and β‐actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The results were visualized using Li‐Cor Odyssey Infrared Imaging System (LI‐COR Biosciences).