2.4. Western Blot Analysis

Western blot was used to determine protein expression of phospho-ERK, ERK, phospho-Akt, Akt, phospho-JNK, JNK, xCT, GLT-1, mGluR5, and β-tubulin in the NAc (core and shell), AMY, and dmPFC. Samples were lysed using a lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1% Triton, 0.1% SDS) with phosphatase and protease inhibitors. The amount of protein in each tissue sample was quantified using a detergent compatible protein assay (Bio-Rad, Hercules, CA, USA). An equal amount of protein from each sample was mixed with laemmili dye, and the mixtures were loaded onto 10% Tris-glycerine gel to separate the protein using electrophoresis. Then, proteins were transferred from gels to a polyvinylidene difluoride (PVDF) membrane. Subsequently, the PVDF membranes were blocked with 5% fat-free milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 30 min. Membranes were incubated overnight at 4 °C with primary antibodies: rabbit anti-p-ERK (1:1000, Abcam, Cambridge, UK, ab201015), rabbit anti-ERK (1:1000, Abcam, ab17942), rabbit anti-p-JNK (1:1000, Cell Signaling, Danvers, MA, USA, 9251), rabbit anti-JNK (1:1000, Cell Signaling, 9252), rabbit anti-p-Akt (1:1000, Cell Signaling, 4060), rabbit anti-Akt (1:1000, Cell Signaling, 4691), rabbit anti-GLT-1 (1:5000, Abcam ab205248), rabbit anti-xCT (1:1000, Abcam ab125186), and rabbit anti-mGluR5 (1:1000, Abcam ab76316). Mouse anti-β-tubulin (1:1000, BioLeagend, San Diego, CA, USA) was used as a control loading protein. On the following day, membranes were washed five times with TBST and incubated with the match secondary antibody (1:4000) for 60 min. The membranes were then washed with TBST and dried for further analysis. The dried membranes were incubated with chemiluminescent reagents (Super Signal West Pico, Perce Inc., Appleton, WI, USA) for 1–2 min. Digitized blot images were developed using the GeneSys imaging system. Quantification and analysis of the expression of p-ERK, ERK, p-JNK, JNK, p-Akt, Akt, GLT-1, xCT, mGluR5, and β-tubulin blots were performed using ImageJ software (Version 1.53t 24). The control group was reported as 100% to measure the changes in the expression of proteins of interest in the NAc (core and shell), AMY, and dmPFC as described in our previous studies [23,29].