Primary antibody against total akt

To assess the mTOR pathway, the phosphorylated AKT protein was first detected using a rabbit polyclonal primary antibody against total AKT (Cell Signaling Technology, Danvers, MA), and HRP-conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA). After detection, the blot was stripped for detection of total AKT (Cell Signaling Technology, Danvers, MA). The data were expressed as the ratio between band intensity of phosphorylated AKT (pAKT) to total AKT.

Western blotting assay was performed as described previously15 (link)27 (link). For the detection of phospho-Akt, the samples prepared in the same day were used. The polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) was incubated with primary antibody against phospho-Akt (Ser473) (Cell Signaling Technology, Beverly, MA), Akt (Cell Signaling Technology, Beverly, MA), phospho-p38-MAPK (Cell Signaling Technology, Beverly, MA), p38-MAPK (Cell Signaling Technology, Beverly, MA), β-actin (Santa Cruz Biotech), GluN2A (Santa Cruz Biotech), or GluN2B (Santa Cruz Biotech). Primary antibodies were labeled with horseradish peroxidase-conjugated secondary antibody, and protein bands were imaged using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA). The EC3 Imaging System (UVP, LLC, Upland, CA) was used to obtained blot images directly from the polyvinylidene difluoride membrane. For the detection of total Akt, the same polyvinylidene difluoride membrane was stripped and then re-incubated with primary antibody against total Akt (Cell Signaling Technology). The quantification of Western blot data was performed using ImageJ software.